Cytokine and hemopoietic factor endogenous production enhancer and methods of use thereof

ABSTRACT

A method of stimulating endogenous production of cytokines and hemopoietic factors by introducing to a mammalian body in need of stimulation of cytokines or hemopoietic factors or both, an effective amount of oxidized glutathione and/or its therapeutically beneficial salts, and/or its therapeutically beneficial derivatives, for a period of time to stimulate said endogenous production to obtain a therapeutic effect. Oxidized glutathione with or without extenders are used in drug forms.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.08/766,557, filed, Dec. 11, 1996, and now issued as U.S. Pat. No.6,251,857, issued on Jun. 26, 2001, which is a continuation-in-part ofU.S. patent application Ser. No. 08/733,886, filed Oct. 18, 1996, andnow issued as U.S. Pat. No. 6,156,979, issued on Dec. 26, 2000.

FIELD OF THE INVENTION

The present invention relates to medicine and more particularly topharmacology and therapy, and is intended to be used for preventing andtreating various diseases by way of increasing endogenous production ofcytokines and hemopoietic factors.

BACKGROUND OF THE INVENTION

It has been known that a number of endogenously produced mammalianhumoral factors, i.e. cytokines and hernopoietic factors possessimportant biological activities that are considerably helpful intreating various human diseases^(1,2). Many of these factors are beingtested in man, those with proven efficiency being commercially availableas medicinal agents.

The following cytokines and hemopoietic factors are being mostextensively researched in oncology: interleukin 2 (IL-2)^(3,4), tumornecrosis factor alpha (TNF-α)⁵, erythropoietin, macrophage-granulocyteand granulocyte colony-stimulating factors (GM-CSF and G-CSF,respectively^(6,7)). No less actively is being studied the use ofcytokines and hemopoietic factors for the treatment of infectiousdisease: interferons (IFN-γ and IFN-β)^(8,9,10), colony-stimulatingfactors^(11,12), and the like¹³. Colony-stimulating factors anderythropoietin are broadly used in hematology^(14,15).

However, the medicinal use of these exogenously administered agents hasits limitations associated with the lack of acceptable drug formulationsor their exorbitant cost, a short half-life of these substances inbiological media, difficulties in dose finding as well as numerous toxicand allergic effects^(16,17), since even the recombinant products aremore or less immunogenic to the human organism because of the processingfluctuations in the course of the artificial synthesis.

In this regard, in view of achieving a more invariable and significanttherapeutic effect free of adverse reactions, it is preferable to inducethe endogenous production of the autologous cytokines and hemopoieticfactors immediately within the organism of a subject. The remedialeffect due to such intrinsic stimulation is free of all thedisadvantages associated with exogenously introduced cytokines andhemopoietic factors.

A number of compounds are currently being evaluated that stimulateendogenous production of cytokines and hemopoietic factors in bothexperimental and clinical settings. There are universally known cases,including successful ones, of using microbial products for cancertherapy which in recent decades has been shown to be mediated viastimulation of the tumor necrosis factor endogenous production¹⁸. Theproducts capable of evoking concomitant production of various cytokinesand hemopoietic factors have presently come to be known asmulti-cytokine inducers. Among these are a killed streptococcalpreparation, Nocardia Opaca, and other bacterial products^(19,20,21).However, virtually all the substances possessing such capability areeither killed microorganisms or microbial products or compounds havingirregular composition, which results in their limited medicinal utilityor even renders their therapeutic use impracticable. Thus, the problemof finding a medically and pharmaceutically acceptable inducer of thecytokine and hemopoietic factor endogenous production has not heretoforebeen resolved.

Oxidized glutathione (also known as glutathione disulfide and GSSG) willoften be referred to as GSSG in this application.

GSSG is known as a dimmer of tripeptide glutathione(γ-glutamyl-cysteinyl-glycine) where two molecules of the tripeptidewith the above structure are linked via a covalent disulfide bondbetween the cystamine residues. Therefore, both the tripeptideglutathione (glutathione, reduced glutathione, GSH; hereinafter referredto as GSH) and its dirmuer GSSG are natural metabolites present inanimal tissues and biological fluids. At the same time, the naturalblood level of GSSG is not sufficient for inducing the cytokineendogenous production in both normal and pathological conditions.

GSH is known to be one of the most important intermediates in the aminoacid metabolism and a factor maintaining the intracellularhomeostasis^(22,23). The reducing properties of GSH and its function asa donor of reduction equivalents, which is due to the sulfhydryl moietyof the cystamine residue, are of key importance. This characteristic ofGSH is responsible for the substance playing a crucial part in one ofthe most important intracellular antioxidant systems, consisting of GSHas such and two enzymes of its reversible conversion into GSSG:glutathione peroxidase and glutathione reductase^(24,25). The permanentfunctioning of said system is essential for inactivating or reducingendogenously generated oxidants as well as active metabolites of foreignsubstances^(26,27).

GSH is also known to participate in detoxification reactions involving agroup of enzymes collectively known as glutathione S-transferase²⁸.These enzymes are capable of conjugating the GSH molecule with variousxenobiotics by forming a bond between the latter and glutathione via thethiol moiety of the cystamine residue of the tripeptide. The subsequentdegradation of the conjugate is catalyzed by the γ-glutamyl cycleenzymes, and may vary considerably depending upon the nature of thexenobiotic.

Under natural conditions, GSSG does not accumulate in amounts sufficientfor inducing cytokine and hemopoietic factor production, due to aconstant reduction of GSSG to GSH. The GSSG reduction to GSH alsoactively progresses in the intestines and liver upon GSSG oraladministration, and as any product made of amino acids, the substance isproteolytically degradable in the gastrointestinal tract.

GSSG is known to be used as a components of a nutritional supplementutilized as an adjunct diet in treating patients²⁹. However, being apeptide substance, most of the orally administered GSSG is digested inthe gastrointestinal tract with the remainder being reduced in theintestinal and hepatic cells to GSH and not entering the circulation.Therefore, the delivery of GSSG into the organism through thegastrointestinal tract may eliminate the possibility of the realizationof its activity as a stimulator of endogenous production of cytokinesand hemopoietic factors.

An elevation of the GSH endogenous levels for medicinal utility is knownto be suggested for boosting immunity³⁰ and treating toxemias,poisonings, diabetes, mellitus, cardiovascular, infectious and otherdisorders^(31,32,33). Possible functions of GSH and GSSG appear in theliterature.

Exogenous GSH or its direct (γ-glutamyl-cystamine, n-acetyl-cystamine,and n-acetyl-cystamine-glycine) or indirect(2-oxothiazolidine-4-carboxylate) biochemical precursors, or their saltsand esters, are reportedly used as medicinal agents and dietarysupplements in treating various diseases^(34,35,36,37,38).

GSH is also claimed to be useful as a chemoprotective agent thatprevents neurotoxicity in cancer chemotherapy³⁹ as well as incombination with antineoplastics in order to augment their effect⁴⁰.

No reference, however, is currently available to GSSG as a medicine inits own right (sole substance) used to induce the endogenous productionof cytokines and hemopoietic factors. The substance is known neither tohave medicinal effects in human and animal diseases nor to be applied asa pharmaceutical agent for treating illnesses.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide an active substance,and advantageous combinations of said substance and/or its derivativeswith extenders and/or enhancers or modulators of its activity which arecapable of inducing endogenous cytokine and hemopoietic factorproduction to an individual or a subject in need thereof.

“Subject in need thereof” as used in this application is intended tomean a mammal, e.g., man, domestic animals and livestock including cats,dogs, cattle and horses, having one or more manifestations of a diseasein which stimulation of endogenous cytokine or hemopoietic factor (orboth) production would be considered beneficial by those skilled in theart. “Therapeutic agent” as used in this application is meant to includeany drug form of GSSG-containing material or GSSG alone, which has atherapeutic effect on neoplastic, infectious, hematologic, immunologicor other diseases. Therapeutic effect, as will be further defined,indicates any effect in man and other mammals which is beneficial,including curative, preventative, allowing maintenance at a beneficiallevel, or is in any way advantageous in connection with the body of manand other mammals.

In accordance with the present invention, it is GSSG that uponparenteral administration induces the endogenous cytokine and/orhematopoietic factor production in an individual or subject in needthereof, in both health and disease.

Having performed studies in search for a medically and pharmaceuticallyacceptable inducer of the cytokine and hemopoietic factor endogenousproduction, the applicants discovered a new property of a previouslyknown substance, oxidized glutathione (oxidized glutathione, glutathionedisulfide, GSSG; hereinafter often referred to as GSSG).

Being administered parenterally or acting on isolated cells, thesubstance is capable of inducing production of several cytokines andhemopoietic factors in mammals (animals and humans) in both health anddisease.

The inducer or stimulator of the endogenous cytokine and hemopoieticfactor production is oxidized glutathione (GSSG) which is a dimmer ofreduced glutathione having the structure γ-glutamyl-cysteinyl-glycine,where the two molecules of the tripeptide are linked via a covalentdisulfide bond between the cystamine residues.

According to the invention, a method is provided for stimulatingendogenous production of cytokine and hemopoietic factors by introducingto a mammalian body in need of stimulation of cytokine or hemopoieticfactor or both, an effective amount of oxidized glutathione for asufficient period of time to stimulate said endogenous production toobtain a therapeutic effect.

Preferably, the glutathione is introduced parenterally or topically. Ina preferred form, the method is carried out by introducing the oxidizedglutathione (GSSG) or its derivatives with an extender of half lifeand/or enhancers or modulators to enhance the desired effect ofstimulating endogenous production of cytokines and hemopoietic factorsand producing a therapeutic effect in a body.

Preferably, the GSSG derivative is selected from the group of compoundsrepresenting a molecule of GSSG chemically modified by bindingcovalently as for example: with cysteamine-(2-mercaptoethylamine),lipoic acid (6,8-thioctic acid), camosine (b-alanyl-hystidine),adenosine (9-β-D-ribofuranosyladenine), methionine(2-amino-4-[methylthio]butanoic acid); and both the D and L forms of theamino acids set forth in this paragraph can be used.

Particularly desirable derivatives are GSSG covalently bound either tocysteamine (S-thioethylamine-glutathione disulfide), or to lipoic acid(bis-[6,8-thiooktanil]•glutathione disulfide), or to camosine([b-alanyl-hystidil]•glutathione disulfide), or to adenosine([9β-D-ribofuranosyladenil]•glutathione disulfide), or to methionine(bis-[2-amino-4-[methylthio]butanoil]•glutathione disulfide), ormixtures thereof and including the D and/or L forms of amino acidsherein.

Preferably, the extender is selected from the group consisting ofpharmaceutically acceptable pro-oxidant compounds, (hydrogen peroxide,ascorbic acid) compounds capable of forming both weak ionic andcoordinating links which stabilize molecule of GSSG (dimethylsulfoxide), or materials which are competitors of NADP-H-dependentreduction of GSSG into GSH catalyzed by glutathione reductase, compoundscapable of producing reversible inhibition of reduction of NADP+ intoNADP-H catalyzed by glucose-6-phosphate-dehydrogenase or by otherNADP-H-dependent enzymes, or mixtures thereof.

Particularly desirable extenders are hydrogen peroxide, inosine,ascorbic acid, dimethyl sulfoxide, or cystamine or mixtures thereof.

Preferably, the enhancer/modulator is selected from the group consistingof methyl moiety donators (such ascholine-chloride{[2-hydroxyethyl]trimethylammonium chloride} orS-adenosyl-methionine), representatives of intracellular redox-oxidativepairs (such as lipoic/dehydrolipoic, folic/dehydrofolic,ascorbic/dehydroascorbic acids). An enhancer or modulator orenhancer/modulator as used herein is meant to be a material whichincreases or changes beneficially in terms of curative outcomes thetherapeutic effect of GSSG or its derivatives, but is not an extender ofhalf life of the GSSG.

Particularly desirable enhancers or modulators are choline-chloride,S-adenosylmethionine, lipoic (6,8-thioctic) and folic (pteroylglutamic)acids.

In the preferred form, GSSG is introduced to the body at a dose of from0.01 to 0.5 mg of GSSG base per kg of body weight for GSSG base and itssalts, and from 0.01 to 1.0 mg for GSSG derivatives, at least one timeduring each 24 hour period, although it can be continuously injected orotherwise introduced to the body to have a 24 hour total dosage of from0.01 to 0.5 mg per kg of body weight for GSSG base and its salts, andfrom 0.01 to 1.0 mg for GSSG derivatives each 24 hour period.Preferably, administration and introduction to the body is carried outuntil a desired stimulating effect increasing production of cytokinesand hemopoietic factors and providing a therapeutic effect is obtained.

According to the invention, a therapeutic agent for treating neoplastic,infectious, hematologic, immunologic and other diseases is provided,comprising an effective amount of oxidized glutathione, along with apharmaceutically acceptable excipient. Preferably, the oxidizedglutathione for parenteral use is in a pharmaceutically acceptablesolvent as, for example, an aqueous solution including water, glucosesolution, isotonic solutions of sodium chloride, buffered saltsolutions. Preferably, a pharmaceutically acceptable extender capable ofenhancing and prolonging therapeutic effect as by increasing the halflife of oxidized glutathione; or a pharmaceutically acceptable enhanceror modulator of GSSG activity by mechanisms other than increasing theGSSG half life, is used along with the GSSG.

The applicants have for the first time shown that an immediate action ofexogenous GSSG or its salts on mammalian (human and laboratory animal)cells capable of producing cytokines and hemopoietic factors, exertsstimulation on the synthesis of these molecules and their increasedlevel in the blood serum (in vivo conditions) or culture media (ex vivoor in vitro conditions). The method suggested can bring about the effectof stimulating production of cytokines and hemopoietic factors, and thiseffect is elicited by the administration of GSSG into the organism orentering into the cultural media, as well as by the administration ofGSSG in combination with pharmacologically active formulations mediatingeither the prolongation of glutathione's retaining the oxidized form orenhancing or beneficially modulating its activity. The studies performedby the applicants have revealed GSSG and its formulations to possess atherapeutic effect in various experimental and clinical pathologicalconditions.

The revealed GSSG-induced stimulation of the endogenous cytokine andhemopoietic factor production in the body results in antitumor,anti-infective, hemopoietic, immunomodulatory and other pharmacologicaleffects resulting, in turn, to a greater or lesser extent therapeutic orpreventive effect in various diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and advantages of the presentinvention will be better understood from the following specificationwhen read in connection with the accompanying drawing in which:

FIGS. 1a, 1 b, 1 c and 1 d are charts showing cytofluorometric analysisof cells HL-60, cytofluorometric analysis of cells HL-60 in the presenceof the preparation of this invention, cytofluorometric analysis of humanlymphocytes, and cytofluorometric analysis of lymphocytes in thepresence of the preparation of this invention, respectively, as will bedescribed in the discussion of Example 4, relating to research ofapoptosis-induced preparation activity in cultivated mammalian cells;and

FIG. 2 is a drawing of GSSG structure with notification of sites forchemical modifications when GSSG salts and derivatives are reproduced;and

FIGS. 3, 4, 5, 6 and 7 are drawings of compounds where GSSG iscovalently bound to: to cysteamine (S-thioethylamine-glutathionedisulfide, FIG. 3); lipoic acid (bis-[6,8-thioktanil]•glutathionedisulfide, FIG. 4); carnosine ([b-alanyl-hystidil]•glutathionedisulfide, FIG. 5), or to adenosine([9β-D-ribofuranosyladenil]•glutathione disulfide, FIG. 6); methionine(bis-[2-amino-4-[methylthio]butanoil]•glutathione disulfide, FIG. 7)

DESCRIPTION OF PREFERRED EMBODIMENTS

In accordance with the present invention, the medicinal agent suggestedfor treating neoplastic, infectious, hematologic, and other diseases, inwhich stimulation of the endogenous cytokine and hemopoietic factorproduction is appropriate, has an effective amount of GSSG and/or itspharmaceutically acceptable salts, and/or its pharmaceuticallyacceptable derivatives as its active principle. It is also advantageousto prepare a drug form of the medicinal agent as an injectable solutioncontaining 0.01 to 2.0% of GSSG base for GSSG itself and its salts, or0.01 to 4.0% for GSSG derivatives.

The GSSG used as a therapeutic or medicinal agent in accordance with thepresent invention is shown in FIG. 2. GSSG and/or its pharmaceuticallyacceptable salts, and/or its pharmaceutically acceptable derivatives ispreferably used in a carrier or solution as, for example, isotonicsolution of sodium chloride, glucose solution, other buffer and saltsolutions. Any aqueous based or solvent based carrier or solvent can beused as long as the overall solution or dispersion is compatible withthe body and pharmaceutically acceptable i.e., it does not cause anyunwanted side effects in the body or unwanted interaction with GSSGand/or its pharmaceutically acceptable salts, and/or itspharmaceutically acceptable derivatives.

In the structural formula of FIG. 2, points X₁, X₂, X₃, X₄, X₅, and X₆are noted as sites for chemical modification of the GSSG. Generally, theGSSG and/or its pharmaceutically acceptable salts, and/or itspharmaceutically acceptable derivatives is used in the form shown insolution or can be any of its physiologically and pharmaceuticallyacceptable soluble salts. The disodium and dilithium salts where X₁, X₄,are either sodium ions or lithium ions or a mixture, are preferred forbest solubility of the drug. X₁, X₂, X₃, and X₄ can each be hydrogen ifother substitutes are not used. Other salts of GSSG can be used, so longas they are pharmaceutically acceptable, i.e., do not adversely affectthe body, for example, X₁, X₂, X₃, and X₄ can all be (or one or more ofthem can be) potassium, calcium, zinc, molybdenum, vanadium, fluoride,mixtures thereof or any other pharmaceutically acceptable substitutes.Water soluble salts are preferred for use in this invention.

In the structural formula of FIG. 3, points X₁ as shown, (or X₁, and X₂)is noted as a site for covalent binding with cysteamine molecule(s)(2-mercaptoethylamine). In the structural formula of FIG. 4, points X₅as shown or X₅, and X₆ are noted as sites for covalent binding withmolecules of lipoic acid (6,8-thioctic acid). In the structural formulaof FIG. 5, point X₃ is noted as a site for covalent binding withmolecule of carnosine (b-alanyl-hystidine). In the structural formula ofFIG. 6, point X₂ is noted as site for covalent binding with molecule ofadenosine (9-β-D-ribofuranosyladenine). In the structural formula ofFIG. 7, points X₅ as shown or X₅, and X₆ are noted as sites for covalentbinding with molecules of methionine (2-amino-4-[methylthio]butanoicacid).

In accordance with the present invention, it is expedient to use suchGSSG or its derivative drug forms and/or pharmaceutical compositionsthat either prolong oxidized glutathione half-life in tissues andbiological fluids, or augment, or beneficially modulate the revealedbiological and therapeutic properties of GSSG.

In accordance with the present invention, with the purpose ofaugmenting, beneficial modulating, and/or prolonging the therapeuticeffect of GSSG, its drug form (injectable solution) is suggested tocontain GSSG and/or its derivatives as described above (see FIGS. 3-7)together with pharmaceutically acceptable component (extender,enhancer/modulator), capable of extending the half-life of GSSG and/orits derivatives or enhancing/modulating their biological andtherapeutical effects. GSSG and/or its salts, and/or its derivatives caneither be present in a single drug form together with the abovementioned extenders, enhancers/modulators (single injectable solutionprepared beforehand or ex tempore), or be delivered into the body usingseparate drug forms: injectable solutions for GSSG and/or its salts,and/or its derivatives and/or derivatives salts; and anypharmaceutically acceptable drug forms, dosage regimens, andadministration routes for the above mentioned extenders,enhancers/beneficial modulators.

As a pharmaceutically acceptable GSSG derivative, one of the compounds,or of their pharmaceutically acceptable salts, where GSSG is covalentlybound to: either cysteamine (S-thioethylamine-glutathione disulfide, seeFIG. 3 for structural formula), or lipoic acid(bis-[6,8-thiooktanil]•glutathione disulfide, see FIG. 4), or carnosine([b-alanyl-hystidil]•glutathione disulfide, see FIG. 5), or adenosine([9β-D-ribofuranosyladenil]•glutathione disulfide, see FIG. 6), ormethionine (bis-[2-amino-4-[methylthio]butanoyl]•glutathione disulfide,see FIG. 7), can be offered for application.

This is because the presence of one of the aforementioned molecules(cysteamine, lipoic acid, carnosine, adenosine, or methionine) as aconstituent part of a modified GSSG molecule, stabilizes structure ofthe corresponding derivative making it more resistant againstproteolysis and/or reduction to GSH. As another way of stabilizingmolecule of GSSG, its salts, or its derivatives/derivative salts, andprotecting them against proteolysis and/or reduction, a replacement ofone or more of L-amino-acids constituting the molecule of both GSSG andthe aforementioned derivatives with their D-forms, can be implemented.

All of pharmaceutically acceptable GSSG or derivatives most preferablycan be used as the medicinal agents in the injectable form of 1.0%solution with dosage range of from 0.01 to 0.5 mg of GSSG base per kg ofbody weight for GSSG base and its salts, and from 0.01 to 1.0 mg forGSSG derivatives, with preferable concentration range of from 0.5% to5.0% one or more times a day, by one or more day pulses or continuouslyuntil a desired therapeutic effect has been achieved.

As a pharmaceutical acceptable component or extender to prolongglutathione permanence in oxidized form, 0.003% hydrogen peroxide and/or5.0% ascorbic acid can be offered for application. This is because inthe presence of hydrogen peroxide or ascorbic acid, a donor of reactiveoxygen intermediates (that is an oxidant), GSSG is reduced byglutathione reductase to GSH at a lesser speed, thereby conditioning aslower reduction of GSSG introduced exogenously into biological media.

Hydrogen peroxide preferably can be used in amounts of from 0.03 to0.0003% by weight of solutions used (from 1.0 to 5.0 ml of solutions,regardless whether they contain or do not contain GSSG and/or its salts,and/or its derivatives/derivative salts). Ascorbic acid preferably canbe used in amounts of from 0.1 to 10% by weight of solutions used (from1.0 to 10.0 ml of solutions, regardless whether they contain or do notcontain GSSG and/or its salts, and/or its derivatives/derivative salts).

Usage of an acceptable concentration of hydrogen peroxide (H₂O₂) and/orascorbic acid in formulation of the drug form for parenteraladministration, as well as usage of any other prooxidant compounds(donors of active oxygen form), makes it possible to realize only one ofpossible methods of the prolongation of oxidized glutathione and/or itsderivative half-life in the biological fluids and tissues and, thereby,to enhance and prolong the pharmaceutical effect of GSSG and/or itsderivatives.

We have also found some other pharmaceutically acceptable components orextenders capable of mediating the slowdown of the reduction ofexogenous GSSG and/or its derivatives into GSH in biological media.Such, in particular, are: the compounds capable of forming weak ionicand/or coordinating links which stabilize molecules of GSSG, forexample, dimethyl sulfoxide; the factors capable of setting upcompetitive relations with a reduced form of the nicotinamide adeninedinucleotide phosphate or NADP-H, for example, inosine (and otherderivatives of hypoxanthine); as well as the agents reversiblyinhibiting the processes of reduction of the oxidized form of NADP+ intoNADPH, for example, cystamine (2,2′-Dithio-bis[ethylamine]) and otherinhibitors of glucose-6-phosphate-dehydrogenase.

Besides hydrogen peroxide and ascorbic acid, one of otherpharmacologically accepted components capable to prolong the oxidizedglutathione half-life can be dimethyl sulfoxide, which stabilize GSSG orits derivative molecules by forming both weak ionic and coordinatinglinks with atoms of GSSG. Dimethyl sulfoxide is used most preferably as7.0% (v/v) solution and preferably as a solution of from 0.1% to 30% byvolume (from 1.0 to 30.0 ml of solutions or more when appliedepicutaneously/through instillations, regardless whether they contain ordo not contain GSSG/GSSG salts and/or its derivatives/derivative salts.

Since reduced NADP-H is the key cofactor of glutathione reductase systemcatalyzing the reduction of GSSG into GSH, any pharmaceuticallyacceptable compounds or biophysical influence retarding the reduction ofGSSG or blocking biological oxidation of NADP-H by glutathione reductasewill facilitate preservation of GSSG/GSSG salts and/or itsderivatives/derivative salts from reduction in biological media and,therefore, will enhance and prolong its curative effect.

Due to conducted research we were the first to discover that GSSGpharmaceutical and medicinal effect will reinforce, when GSSG used incombination with agents capable of competition with NADP-H, as well aswith compounds reversibly inhibiting the enzymatic reaction, catalyzedby glucose-6-phosphate-dehydrogenase which mediates the reduction of theoxidized form of NADP+. Reversible inhibitors or pentose phosphatepathway of glucose oxidation can be used.

Thus, besides hydrogen peroxide, ascorbic acid and dimethyl sulfide oneof other pharmacologically accepted components capable to prolong theoxidized glutathione half-life can be inosine(hypoxanthine-9-D-ribofuranoside) used most preferably as 0.1% solutionand preferably as a solution of from 0.1% to 5% by weight (from 1.0 to5.0 ml of solutions, regardless whether they contain GSSG/GSSG saltsand/or its derivatives/derivative salts.

The investigations carried out showed inosine to facilitate biologicaland therapeutical effects of GSSG. It was demonstrated that thisproperty of inosine is based on its ability to compete with NADP-H, andthereby, to retard GSSG reduction into GSH. Moreover, we have also foundthat other hypoxanthine derivatives (including inosine, nucleoside ones,hypoxanthine riboside and other nucleoside derivatives of inosine)possess this property as well.

Also, besides hydrogen peroxide, ascorbic acid, dimethyl sulfoxide andinosine, cystamine (2,2′-Dithio-bis[ethylamine]) is anotherpharmaceutically acceptable agent conditioning a slower reduction ofGSSG, if used most preferably as 0.1% solution and preferably as asolution of from 0.1% to 3% by weight (for example 1.0 to 5.0 ml ofsolutions, regardless of whether they contain GSSG/GSSG salts and/or itsderivatives/derivatives salts).

Our research showed cystamine to facilitate biological and therapeuticaleffects of GSSG. The effect is due to the cystamine ability to act as areversible inhibitor of key enzyme of the pentose phosphate pathway,glucose-6-phosphate-dehydrogenase, mediating reduction of NADP+ intoNADP-H.

As pharmaceutically acceptable components capable of enhancing orbeneficially modulating biological and therapeutic effects of GSSGand/or its derivatives, several groups of chemical compounds have beenshown to augment, diversify or beneficially alter effects of GSSG and/orits derivatives. Therefore several enhancers/beneficial modulators ofeffects of GSSG/GSSG salts and/or its derivatives/derivative salts canbe assigned to the following groups of chemicals.

Donators of methyl groups, such as choline-chloride andS-adenosyl-methionine used in combination with GSSG (and/or itssalts/derivatives) have appeared to be more effective compared with GSSGalone (and/or its salts/derivatives) when these agents are used fortreating animals with experimental pathologic conditions of immunologicand infectious nature. At that, it has been shown that choline-chlorinecan be used in patients most preferably as 10% solution and preferablyas a solution of from 1.0% to 20% by weight (from 1.0 to 5.0 ml orsolutions, regardless whether they contain GSSG or its derivatives).S-adenosyl-methionine can be used in patients most preferably as 5.0%solution and preferably as a solution of from 1.0% to 10% by weight(from 1.0 to 5.0 ml of solutions, regardless whether they contain eitherGSSG and/or its derivatives).

Compounds, which are capable of formation intracellular redox-oxidativepairs (lipoic, folic and ascorbic acids) have also been found to augmentGSSG/derivative effects in immunologic, infectious, or other diseases(diabetes mellitus). Lipoic acid can be used in patients most preferablyas 0.5% solution and preferably as a solution of from 0.1% to 1.0% byweight (from 1.0 to 5.0 ml of solutions, regardless whether they containeither GSSG and/or its derivatives). Folic acid can be used in patientsmost preferably as 0.5% solution and preferably as a solution of from0.1% to 1.0% by weight (from 2.0 to 5.0 ml of solutions, regardlesswhether they contain either GSSG and/or its derivatives).

Thus, the present invention also suggests the method to enhance orbeneficially modulate the ability of GSSG to stimulate endogenousproduction of cytokines and hemopoietic factor which presupposes theusage a pharmaceutical composition including GSSG and/or its derivativesand an additional component or components able to prolong the oxidizedglutathione half-life (extenders) or to enhance/modulate beneficiallybiological and therapeutical effects of GSSG and/or its derivatives(enhancers/beneficial modulators). GSSG and/or its salts, and/or itsderivatives can either be administered combined in a single dosage formwith both extenders and enhancers/modulators, or can be delivered into abody separately from both extenders and enhancers/modulators, usingdifferent pharmaceutically acceptable administration routes for eachconstituent of any combination used. This can be achieved for example bythe administration of pharmaceutically acceptable compositions includingdrug forms of GSSG/GSSG salts and/or GSSG derivatives/derivative salts;and pharmaceutically acceptable compositions including drug forms ofother products, able to prolong the oxidized glutathione half-life(extenders), and/or able to enhance/modulate beneficially therapeuticaleffects of GSSG/GSSG salts and/or GSSG derivatives/derivative salts.

As used herein, the term “GSSG derivatives” means eitherS-thioethylamine-glutathione disulfide, orbis-[6,8-thiooktanil]•glutathione disulfide, or[b-alanyl-hystidil]•glutathione disulfide, or[9β-D-ribofuranosyladenil]•glutathione disulfide, orbis-[2-amino-4-[methylthio]butanoil]•glutathione disulfide, with one ormore of L-amino-acids constituting the molecule GSSG being replaced withits D-form and given at the same dosage range (of from 0.01 to 1.0mg/kg).

As used herein, the term “extenders” means hydrogen peroxide preferably0.003%, ascorbic acid preferably 5.0% or other compounds with oxidantactivity; dimethyl sulfoxide preferably 7.0%, or other compounds capableof forming weak ionic and/or coordinating links which stabilize moleculeof GSSG; inosine (hypoxanthine-9-D-ribofuranoside) preferably 0.1%, orits derivatives including inosine nucleosides; and also cystamine(2,2′-Ditio-bis[ethylamine] preferably 0.1%, or other compounds, capableto produce reversible inhibition of glucose-6-phosphate-dehydrogenase,the key enzyme of the pentose phosphate pathway.

As used herein, the term “enhancers/beneficial modulators” or“enhancers/modulators” means choline-chloride preferably 10%,S-adenosyl-methiomine preferably 5.0% or other pharmaceuticallyacceptable donators of methyl groups; lipoic acid preferably 0.5%; folicacid preferably 0.5% or other compounds, which are capable of formationintracellular redox-oxidative pairs. Any other chemical compound orphysical influence, which is capable of enhancing and/or modulatingbeneficially any: biological or therapeutical effects ofGSSG/derivatives mentioned in this application should also be consideredas “enhancers/modulators.”

As used herein, the term “epicutaneously/through instillations” meansany physiologically and/or medically acceptable administration routewhen there is a remedy/medicine application on skin surface orsuperficial mucous membrane (epicutaneous, or cutaneous, or topical, orlocal use), or when there is an intracavitary remedy/medicine usethrough its introduction into a natural and/or artificial cavity of (orspace within) a body, such as stomach, urinary bladder, vagina, rectum,abdominal or pleural cavities, intraarticular space, airways, maxillarysinus, and the like, or any pathological, and/or wound cavity.

It is found that the parenteral (intravenous, intraperitoneal,intramuscular, etc.) administration of GSSG and/or its salts, and/or itsderivatives in combination with an extender or enhancer/modulatorstimulates endogenous production or TNF-α, IFN-α and IFN-γ, IFN-B,IL-IB, IL-1, IL-2, IL-6, IL-10, G-CSF, colony stimulating factors,erythropoietin, and GM-CSF in organism of experimental animals in alarger degree than with the application of GSSG alone and/or its salts,and/or its derivatives. GSSG and/or its derivatives and any extender orenhancer/modulator can be administered with the use of both the singledosage form or different pharmaceutically acceptable dosage forms (aswell as dosage regimens and administration routes) for each constituentof any combination used.

The studies carried out prove the ability of the above mentionedcompounds to enhance and/or beneficially alter the biological andtherapeutical effects of GSSG and/or salts and/or derivatives, whichmakes evident the expediency of their use in combination with GSSGand/or salts and/or derivatives, to treat neoplastic, infectious,hematological and other diseases in which stimulation of the endogenouscytokine and hemopoietic factor production is considered beneficial bythose skilled in the art.

Thus, in accordance with the present invention, for the purpose ofenhancing and prolonging the GSSG therapeutical effect, it is preferredthat a final drug formulation of GSSG or its salts orderivatives/derivative salts (1-5 ml of solution for injections) shouldcontain additional pharmaceutically acceptable components able to eitherprolong the GSSG, salt or derivative half-life in the biological mediaor to enhance/modulate beneficially their biological or therapeuticaleffects. Any of proposed pharmaceutically acceptable components can alsobe administered separately from GSSG and/or its derivatives, with theuse of any other pharmaceutically acceptable dosage form, as well asdosage regimen, and route of administration.

These pharmaceutically acceptable components, which are other than GSSGand its derivatives, and their most preferable concentrations anddosages for treating human beings can be the following:

a) 0.003% hydrogen peroxide with the acceptable concentration range offrom 0.03 to 0.0003% by weight and dosage range of from 1.0 to 5.0 mland more when administered epicutaneously or through instillations; 5.0%ascorbic acid with the acceptable concentration range of from 0.1 to 10%by weight and dosage range of from 1.0 to 10.0 ml and more whenadministered epicutaneously or through instillations; or any otherpharmaceutically acceptable pro-oxidant compounds with activity of thedonors of reactive oxygen intermediates;

b) 7.0% (v/v) dimethyl sulfoxide with the acceptable concentration rangeof from 0.1% to 30% by volume and dosage range of from 1.0 to 30.0 mland more when administered epicutaneously or through instillations; anyother pharmaceutically acceptable compounds capable to stabilize GSSG orits derivative molecule by forming both weak ionic and coordinatinglinks with atoms of GSSG;

c) 0.1% inosine (hypoxanthine-9-ribofuranoside) with the acceptableconcentration range of from 0.1% to 5.0% by weight and dosage range offrom 1.0 to 5.0 ml and more when administered epicutaneously or throughinstillations; any other pharmaceutically acceptable competitors ofNADP-H-dependent reduction of GSSG into GSH catalyzed by glutathionereductase;

d) 0.1% cystamine (2,2′-Dithio-bis[ethylamine]) with the acceptableconcentration range of from 0.1% to 3.0% by weight and dosage range offrom 1.0 to 5.0 ml and more when administered epicutaneously or throughinstillations; any other pharmaceutically acceptable compounds able toproduce reversible inhibition or reductio of NADP+ into NADP-H catalyzedby glucose-6-phosphate-dehydrogenase or by other NADP-dependent enzymes.

e) 10% choline-chloride with the acceptable concentration range of from1.0% to 20% by weight and dosage range of from 1.0 to 5.0 ml and morewhen administered epicutaneously or though instillations; 5.0%S-adenosyl-methionine with the acceptable concentration range of from1.0% to 10% by weight and dosage range of from 1.0 to 5.0 ml and morewhen administered epicutaneously or through instillations; any otherpharmaceutically acceptable compounds able to serve as donator of methylgroups;

f) 0.5% lipoic acid with the acceptable concentration range of from 0.1%to 1.0% by weight and dosage range of from 1.0 to 5.0 ml and more whenadministered epicutaneously or through instillations; any otherpharmaceutically acceptable compounds able to form intracellularredox-oxidative pairs.

At the same time, the data were obtained to testify the direct antitumoreffect of GSSG, GSSG salts or GSSG derivatives administered alone or incombination with the pharmaceutically acceptable compounds prolongingoxidized glutathione half-life in biological media orenhancing/modulating the effects thereof. Moreover, the GSSG or GSSGderivative effect was proved to be different for normal and tumor cells.Our in vitro research with the use of normal and tumor cells revealedthat the GSSG or its derivatives alone, or their pharmaceuticallyacceptable compositions containing extenders and/or enhancers/modulatorsinitiated tumor cell death in apoptosis like manner. In case of normalcells, they did not undergo destruction.

It is an object of the present invention to provide a method fortreating neoplastic, infectious, hematologic and other diseases in whichstimulation of the endogenous cytokine and hemopoietic factor productionis advantageous. The method comprises parenteral administration of GSSGand/or its. derivatives as the medicinal agent in the injectable drugform at 0.01 to 0.5 mg of GSSG base per kg body weight or 0.01 to 1.0mg/kg for GSSG derivatives, one or more times a day, by one or more daypulses or continuously until a desired therapeutic effect has beenachieved. It is essential that either GSSG as medicinal agent or itsdrug forms and/or pharmaceutical compositions be administered strictlyparenterally so that to prevent or minimize its deregulation orreduction (to GSH) taking place in the gastrointestinal tract upon oraladministration. However, any of proposed pharmaceutically acceptablecomponents such as extenders, enhancers/modulators can also beadministered separately from GSSG and/or its derivatives, with the useof any other pharmaceutically acceptable dosage form, as well as adosage regimen, and a route of administration. At that, the GSSG and/orits derivatives with or without extenders and/or enhancers/modulatorscan also be applied topically or epicutaneously to the body at a doseconsistent with the parenteral and intercavity dose as for example 0.01to 0.5 mg of GSSG base per square meter of skin or topical areas of thebody being treated (with 0.01 to 1.0 mg per square meter for GSSGderivatives).

Provided the GSSG or its derivative molecule is protected fromproteolysis and/or reduction to GSH, it would be advantageous toadminister the agent orally and/or intralesionally (in situ) (wound,tumor, etc.).

The examples given below confirm that the parenteral (intraperitoneal,intravenous, intramuscular, subcutaneous, etc.) use of GSSG and/or itsderivative results in inducing the endogenous production of inter aliaTNF-α, IFN-α and IFN-γ, IL-1, IL-2, IL-6, IL-10, erythropoietin, andGM-CSF in mammals, which elicits a significant therapeutic effect inanimals and humans suffering from neoplastic or infectious disease,hemopoiesis and immunity suppression of different origin, and otherdiseases in which stimulation of the endogenous cytokine and hemopoieticfactor production would be considered beneficial by those skilled in theart.

From the experimental findings (see Examples) it follows that thepreviously unknown GSSG capability of inducing the endogenous cytokineand hemopoietic factor production and exerting beneficial effects invarious diseases, is not associated with an increase in GSH levels,because GSH testing in a wide range of doses and concentrations hasrevealed neither stimulation of the endogenous cytokine and hemopoieticfactor production nor the therapeutic effect observed with the use ofGSSG and/or its derivatives.

GSSG can be used along with other medicaments without causing unwantedinteraction in the body. For example, patients treated with known drugssuch as lithium, ibuprofen, aminophylline, antibiotics, AZT, calciumantagonists, tamoxifen, hormones, interferon, and others can be treatedsimultaneously with GSSG, its salts and/or derivatives.

As used herein, the term “therapeutic effect” means any improvement inthe condition of a patient or animal treated according to the subjectmethod, including obtaining a preventative or prophylactic effect, orany alleviation of the severity of signs and symptoms of a disease andits sequelae, including those caused by other treatment methods (e.g.,chemo- and X-ray therapy), which can be detected by means of physicalexamination, laboratory or instrumental methods and consideredstatistically and/or clinically significant by those skilled in the art.

As used herein, the term “prophylactic effect” means prevention of anyworsening in the condition of a subject treated according to the methodof the invention, as well as prevention of any exacerbation of theseverity of signs and symptoms of a disease or its sequelae, includingthose caused by other treatment methods (e.g. chemo- and X-ray therapy),which can be detected by means of physical examination, laboratory orinstrumental methods and considered statistically and/or clinicallysignificant by those skilled in the art.

As used herein, the terms “neoplastic and infectious disease”,“hemopoiesis and immunity depression of various origin”, and “otherdiseases” mean any neoplastic and infectious disease, any conditioncaused or accompanied by the erythroid or myeloid suppression, or areduction in quantitative or functional immunity parameters, as well asany other disease or pathological condition in which stimulation of theendogenous cytokine and/or hemopoietic factors including but not limitedto TNF-α, IFN-α, and INF-γ, IL-1, IL-2, IL-6, IL-10, erythropoietin, andGM-CSF, production would be considered advantageous by those skilled inthe art.

The non-limiting examples given below demonstrate feasibility of theinvention.

The active principle, the GSSG peptide capable of inducing theendogenous cytokine and hemopoietic factor production, may be obtainedby conventional peptide synthesis technique⁴¹.

Thereby obtained peptide (GSSG) is subsequently used in animals andhumans (in vivo) as the GSSG base, or as a pharmaceutically acceptableGSSG salt, or as a pharmaceutically acceptable GSSG derivative in aninjectable drug form prepared by dissolving the bulk substance ininjectable water, or in any pharmaceutically acceptable solvent, withthe resultant concentration of the active compound being 0.01-2.0% byweight of GSSG base for GSSG and its salts (with 0.01 to 4.0% by weightfor GSSG derivatives).

For an in vitro use in experimental settings, GSSG or its derivativesmay be dissolved in biologically acceptable solvents such as culturemedia, isotonic saline solutions, glucose solutions and the like.Preferably an aqueous carrier or solvent is used, although andphysiological and other solvents or carrier can be used. For topicalapplication, organic solvents or carriers may be used in the form ofointments, pastes, creams or suppositories for body orificeapplications.

The drug form for human and animal use should be prepared under sterileand pyrogen-free conditions while exerting every effort to preventchemical or bacterial contamination, thereby providing a sterile,pyrogen free treating agent or drug form.

The GSSG or its derivatives injectable drug form has been tested in bothanimal studies and pilot human trials.

The use of the maximum achievable concentration of the GSSG sodium saltsolution (10.0%, 100 mg/mL) in injectable water (or in normal saline, orin 0.003% hydrogen peroxide, or in 0.1% cystamine), and the maximumtolerable volumes administered to mice intra-peritoneally (IP<2.0 mL),intravenously (IV, 0.5 mL), and intramuscularly (IM, 0.05 mL), have madeit feasible to reach GSSG dosage levels 5000 mg/kg (IP), 1350mg/kg (IV),and 135 mg/kg (IM), i.e. 1000, 270, and 27 times, respectively, themaximum recommended human dose of 0.5 mg/kg. In none of the cases eitheranimals' deaths or any toxic signs were observed, showing GSSG ininjectable drug form to be essentially non-toxic.

The results of nonclinical evaluation of biological, pharmacological,and therapeutical properties of GSSG, we well as its drug forms with orwithout 0.003% hydrogen peroxide, or 0.1% inosine, or 0.1% cystamine,are presented in Examples ##1-5.

EXAMPLE #1 Effect of GSSG and its Drug Forms on Cytokine Production byHuman Peripheral Blood Mononuclear Leukocytes in Vitro

Oxidized glutathione (GSSG), as well as its drug forms containing 0.003%hydrogen peroxide, or 0.1% inosine, or 0.1% cystamine, were evaluatedfor their effect on cytokine production by human peripheral bloodmononuclear leukocytes in vitro.

The leukocytic cytokine production was triggered by adding a mitogen,concanavalin A (ConA) to the cell culture immediately after introducingthe test substances. In 24 hours of the cellular exposure to ConA andthe test articles, the culture supernatants were sampled and storeduntil cytokine determination at −70° C.

With the aim of evaluating the functional status of the cells and theircapacity of responding to the mitogen in the presence of the testarticles at each concentration level, the control cell cultures,containing the test articles in identical concentrations, were incubatedfor 72 hours following the initial concomitant introduction of ConA andthe test substances. 16 hours prior to the incubation completion,³H-thymidine was added, and the label rate of incorporation into DNA wasinterpreted as the criterion of the cellular test system functionalstate.

Venous blood from male healthy volunteers was collected into plasticheparinized tubes (endotoxin tested). PMNL fraction was isolated bycentrifugation in density gradient of Ficoll and sodium diatrizoate(Histopaque-1077; Sigma).

Cell concentration was adjusted to 2×10⁶ per mL of “complete” culturemedium (RPMI 1640, Sigma) containing: HEPES (20 mM); L-glutamine (2 mM);Gentamicin (50 μg/mL); fetal calf serum (10%). All the reagents usedwere of “cell culture tested” grade, Sigma. Cell viability was estimatedby the Trypan blue exclusion method and 100 μL of cell suspension(200,000 cells) was placed into each well of flat bottom 96-well sterilemicro titer plates for tissue cultures. Cells from each subject wereplaced into no less than 39 wells.

The five following final concentrations of the test articles (GSSG, aswell as its drug forms containing 0.003% H²O², or 0.1% inosine, or 0.1%cystamine) were evaluated: 5000 μg/mL; 500 μ/mL; 50 μg/mL; 5 μg/mL; and0.5 μg/mL. Each concentration was established in no less than 6 wells byadding 50 μL of “complete” medium containing the appropriate quantity ofthe previously dissolved test articles. Another 6 wells were used forcontrol cultures and contained no GSSG: 50 μL of “complete” medium, orcorrespondingly, “complete” medium containing 0.003% H₂O₂, or 0.1%inosine, or 0.1% cystamine, were added.

Immediately after the test articles had been entered into the cultures,50 μL of “complete” medium containing ConA (Sigma, cell culture tested)in a quantity required for a final concentration of 4.0 μg/mL, was addedto all the wells excepting 3 additional ones which served for evaluationof spontaneous ³H-thymidine uptake (without ConA). After a twenty fourhour incubation at 37° C. and 5% of CO₂, contents of 3 wells (from eachsextuplet of identical wells) were taken out, centrifuged, and thesupernatants were frozen and kept at −70° C. until the cytokine assay.Cultures in the other 3 wells (of each sextuplet were incubated furtherunder the conditions described above. Fifty six hours after theincubation had begun, 1.0 μCi of ³H-thymidine was added into all theremaining cultures, the plates were incubated for another 16 hours, andthen the contents of the wells were harvested and transferred ontoglass-fiber filters which were consequently treated with 5%trichloroacetic acid and ethanol. The filters were dried and theirradioactivity (counts per minute, cpm) was determined using liquidscintillation counter, Betaplate 1205 (LKB).

Mean radioactivity values for triplicates of identical cultures wereused to calculate the index of mitogenic stimulation: the ratio ofaveraged cpm values of ConA stimulated cultures to averaged cpm valuesof unstimulated ones (3 wells without ConA). This stimulation index forwells, where the test articles were present in various concentrations.served as a criterion of cellular functional status, and ability of thecells to respond to mitogenic stimulation.

Supernatants of 24-hour culture triplicates were subsequently assayedfor cytokine content only if their 72-hour matched control culturetriplicates developed mitogenic response to ConA with value of thestimulation index in the range from 15 to 50.

Concentrations of interleukin-1b), interleukin-6 (IL-6), tumor necrosisfactor α (TNFα), and interferon α (IFNα) were determined by ELISA usingcommercial reagent kits (Medgenix, Belgium) and were expressed in pg/mLof culture supernatants. The salient findings given in Tables 1-4. Ascan be seen from Tables 1 and 2, the adding of GSSG into the culturemedia resulted in statistically significant and dose-dependentstimulation of the cytokine production by human mononuclear leukocytes.In addition, the presence of hydrogen peroxide leads to increase control(no GSSG) levels of IL-6 and TNF-α. Besides that, being used incombination with hydrogen peroxide GSSG exerts a more pronounced (1.5-2fold) stimulatory effect on the production of the cytokines on study:for IL-1μ— at 5.0-5000 μg/mL concentration levels; for IL-6 and TNF-α—inthe entire concentration range; and for IFN-α—at 500 and 5000 μg/mL.

The application of GSSG in 0.1% inosine solution and 0.1% cystaminesolution results in a significant and dose-dependent increase ofcytokine production, particularly with respect to IL-6 and TNFα (Tables3 and 4).

Thus, the GSSG effect on the human peripheral blood mononuclearleukocytes in vitro manifests in considerable stimulation of the ctokinerelease into culture media, thereby confirming the stimulatory effect ofGSSG on the natural cytokine-producing capacity of the human bloodcells. The use of GSSG in combination with hydrogen peroxide, inosine,as well as cystamine results in a more prominent effect of GSSG inrespect of induction of endogenous cytokine production.

TABLE 1 GSSG effect on in vitro cytokine production by human mononuclearleukocytes. (M ± m) Cytokine production (pg/mL) GSSG (μg/mL) IL-1β IL-6TNFa IFNa 5000 259 ± 36.8* 2518 ± 264* 1900 ± 206* 511 ± 64.1* 500 275 ±39.3* 2113 ± 132* 1525 ± 163* 514 ± 56.2* 50 202 ± 24.9* 1910 ± 205* 813± 90.8* 407 ± 51.4* 5.0 88.5 ± 13.5* 550 ± 61.3* 314 ± 44.7* 109 ± 12.10.5 56.0 ± 9.1 430* ± 55.6 99.1 ± 11.6 130 ± 14.9 Control (RPMI) 46.0 ±6.8 129 ± 12.4 88.7 ± 9.3 98.3 ± 14.0 *—differences are statisticallysignificant (p < 0.01) as compared to the control.

TABLE 2 Effect of GSSG in combination with 0.003% hydrogen peroxide onin vitro cytokine production by human mononuclear leukocytes. (M ± m)Cytokine production (pg/mL) GSSG (μg/mL) IL-1β IL-6 TNFα IFNα 5000 720 ±81.3* 4035 ± 518* 2640 ± 355* 849 ± 102* 500 650 ± 67.1* 4007 ± 419*2100 ± 294* 905 ± 141* 50 511 ± 55.1* 3859 ± 425* 1308 ± 164* 468 ±69.3* 5.0 212 ± 31.7* 1680 ± 207* 502 ± 86.4 160 ± 37.0 0.5 63.0 ± 7.8851 ± 111 318 ± 47.8 98.3 ± 18.7 Control 51.0 ± 7.4 970 ± 140 410 ± 57.0125 ± 20.8 (RPMI + 0.003% H₂O₂) *—differences are statisticallysignificant (p < 0.01) as compared to the control.

TABLE 3 Effect of GSSG in combination with 0.1% inosine on in vitrocytokine production by human mononuclear leukocytes. (M ± m) Cytokineproduction (pg/mL) GSSG (μg/ml) IL-1β IL-6 TNFα IFNα 5000 665 ± 73.5*5720 ± 498* 5900 ± 317* 1010* ± 160.5* 500 790 ± 68.85* 3840 ± 352* 4520± *366 1318 ± 152* 50 416 ± 44.0* 4910 ± 205* 1869 ± 90.8* 311 ± 51.4*5.0 205.8 ± 18.3* 2680 ± 196* 765 ± 67.1* 117 ± 10.4* 0.5 183 ± 20.0*1505 ± 138* 597 ± 48.6* 66.3 ± 7.8* Control (RPMI + 0.003% 60.9 ± 5.59*131 ± 11.7* 83.5 ± 9.6* 89.5 ± 10.0* H₂O₂)

TABLE 4 Effect of GSSG in combination with 0.1% cystamine on in vitrocytokine production by human mononuclear leukocytes. (M ± m) Cytokineproduction (pg/mL) GSSG (μg/ml) IL-1β IL-6 TNFα IFNα 5000 810 ± 75.36*4910 ± 503* 5140 ± 466* 1060 ± 799* 500 540 ± 60.03* 4000 ± 307* 3800 ±307* 780 ± 180.3* 50 490 ± 45.5* 3800 ± 3183* 2600 ± 183 460 ± 39* 5.0316 ± 30.5* 2610 ± 207* 1408 ± 101* 100 ± 17.7* 0.5 155 ± 9.7* 10 ± 110*709 ± 67.3* 107.6 ± 8.13* Control (RPMI + 0.1% 60.8 ± 6.55* 65.4 ± 77.0*377 ± 28.9* 114 ± 10.01* cystamine) *—differences are statisticallysignificant (p < 0.01) as compared to the control.

EXAMPLE #2 Effect of GSSG and its Drug Forms on Cytokine and HemopoieticFactor Production as Well as on Hemopoiesis and Immunity Parameters inCyclophosphamide-induced hemo- and Immunodepression

Both oxidized (GSSG) and reduced (GSH) glutathione, as well as GSSG drugforms containing 0.003% hydrogen peroxide, or 0.1% inosine, or 0.1%cystamine, were evaluated in a murine model of hemo- andimmunodepression induced by a single administration of cytostaticcyclophosphamide (CP).

The study was designed to evaluate the effect of a 5-day longadministration of the test articles on the capability of the CP-treatedmurine splenocytes to produce IL-2 and GM-CSF in vitro. In addition, thenumber of blood leukocytes and lymphocytes and the bone marrowcellularity (number of karyocytes) were determined at 8 days after CPadministration. Some animals receiving CP were then challenged withsheep red blood cells (SRBC), and the humoral immune response to theantigen was evaluated.

Male CBA mice (18 to 20 g body weight) were given a singleintraperitoneal injection of CP in a dose of 50 mg/kg. Five groups ofanimals (with no less than 15 mice in each) were formed. Groupdescription is represented below.

Control Groups:

#1—intact animals receiving a single injection of normal saline (NS)instead of CP injection, which further were treated with test articlevehicle (normal saline);

#2—control animals receiving a single CP injection, which further weretreated with test article vehicle (normal saline);

#3—animals receiving a single CP injection, which further were treatedwith s reference article (GSH dissolved in normal saline) in a dose of 5mg/kg;

Test Groups:

#4—animals receiving a single CP injection, which further were treatedwith the test article (GSSG dissolved in normal saline) in a dose of 5mg/kg;

#5—animals receiving a single CP injection, which further were treatedwith a variant of the test article drug form (GSSG dissolved in normalsaline containing 0.003% H₂O₂) with a GSSG dose of 5 mg/kg;

#6—animals receiving a single CP injection, which further were treatedwith a variant of the test article drugform (GSSG dissolved in normalsaline containing 0.1% inosine) with a GSSG dose of 5 mg/kg;

#7—animals receiving a single CP injection, which further were treatedwith a variant of the test article drugform (GSSG dissolved in normalsaline containing 0.1% cystamine) with a GSSG dose of 5 mg/kg;

Twenty four hours after the CP injection, 5 animals in each group wereimmunized with SRBC (107 cells in 0.5 mL of NS, intraperitoneally). Onday 3 after the CP injection (24 hours after the immunization) theintraperitoneal injections of the test or reference articles werestarted (as it has been described above). Injections were performedduring 5 days: once a day, daily.

Twenty four hours after the completion of 5 day treatment course (on the8th day after the CP injection), mice were euthanized and splenocytecultures were aseptically prepared for assessment of spontaneousproduction of IL-2 and GM-CSF by the spleen lymphocytes in vitro.

Simultaneously, blood and marrow samples were collected for bloodleukocyte and lymphocyte, and marrow nucleated cell counted,

Serum samples from immunized animals were tested on level of SRBCagglutinins (the day 8 after the CP injection, and the day 7 after theimmunization).

Table #5 shows the parameters of IL-2 and GM-CSF production bysplenocytes, bone marrow and blood cellular indices, and the immuneresponse to sheep red blood cells in mice receiving the test articlesagainst the background of cyclophosphamide induced hemo- andimmunodepression.

As is seen from the data, the use of both GSSG and GSSG solution inhydrogen peroxide brings IL-2 and GM-CSF splenocytic production almostback to normal whereas GSH shows no such effect. Also, both GSSG and itshydrogen peroxide solution exert a significant restorative effect on thebone marrow and blood parameters as well as immune response to SRBC.

Tables ##6 and 7 give data on effects of pharmacologically activecompositions containing GSSG (in combination with 0.1% of inosine, or0.1% cystamine) on tested parameter variations in mice with CP-inducedhemo- and immunodepression. The findings show significant enhancing GSSGeffects by inosine and cystamine components with respect of IL-2b andGM-CSF production stimulation and restoration of bone marrow and bloodcellularity. As it could be seen, GSH did not exhibit such stimulation.The maximum stimulation was achieved with the combination of GSSG and0.1% inosine.

Thus, the use of the subject method in CP-induced hemo- andimmunocompromised animals results in a prominent stimulation of IL-2 andGM-CSF endogenous production together with restoration of the bonemarrow and blood cellular indices as well as immune response developmentto sheep red blood cells.

TABLE 5 Effect of the test articles on IL-2 and GM-CSF production bysplenocytes, bone marrow and blood cellular indices, and immune responseto SRBC in cyclophosphamide treated mice. (M ± m) Intact animalsCyclophosphamide-treated animals Normal Normal GSSGO + Parameter nsaline saline GSH GSSG H₂O₂ IL-2 production 10 39.7 ± 5.4 11.1 ± 3.0*17.2 ± 3.5* 28.1 ± 3.9^(#@) 34.7 ± 51.^(#@) by splenocytes, (U/mL)GM-CSF 10 180.0 ± 14.2 34.3 ± 9.1* 58.2 ± 7.2* 129.1 ± 13.4^(#@) 170.1 ±16.9^(#@) production by splenocytes, (colonies/10⁵ cells) Blood 10 11.9± 1.81 4.7 ± 1.25* 5.2 ± 1.36* 8.5 ± 0.18^(#@) 9.4 ± 1.40^(#@) leukocytecount, 10⁹/L Blood 10 7.4 ± 0.85 3.1 ± 0.56* 4.3 ± 1.13* 6.2 ± 1.28^(#)6.8 ± 1.04* lymphocyte count, 10⁹/L Bone marrow 10 53.7 ± 8.7 23.8 ±5.0* 32.2 ± 4.4* 45.4 ± 3.9^(#@) 52.3 ± 4.7^(#@) nucleated cell number,10⁶/L SRBC  5 5.33 ± 0.74 1.47 ± 0.35* 1.94 ± 0.34* 3.68 ± 0.59*^(#)4.12 ± 0.37*^(#) agglutinin titer (log₂) Differences are statisticallysignificant (p < 0.05) as compared: *—to the group of intact animals;^(#)—to the controlg roup (CP + normal saline); ^(@)—to the group ofanimals treated with GSH.

TABLE 6 Effect of GSSG in combination with 0.1% inosine on IL-2 andGM-CSF production by splenocytes, bone marrow and blood cellularindices, and immune response to SRBC in cyclophosphamide treated mice.(M ± m) Intact animals Cyclophosphamide-treated animals Normal NormalGSSG + Parameter n saline saline GSH GSSG 0.1% inosine IL-2 productionby 10 34.4 ± 4.2 9.2 ± 1.9* 15.3 ± 2.7* 2.8 ± 3.158^(#@) 39.7 ± 4.8^(#@)splenocytes, (U/mL) GM-CSF production by 10 168.0 ± 14.9 25.5 ± 4.2*63.4 ± 7.8* 143 ± 15.06^(#@) 196.3 ± 16.6^(#@) splenocytes,(colonies/10⁵ cells) Blood leukocyte count, 10 123 ± 14 5.03 ± 0.85* 6.3± 0.05* 9.5 ± 1.01^(#@) 10.1 ± 1.36^(#@) 10⁹/L Blood lymphocyte count,10 8.2 ± 0.09 2.8 ± 0.67* 4.6 ± 0.78* 6.7 ± 0.81^(#) 7.18 ± 0.74^(#)10⁹/L Bone marrow nucleated 10 61.3 ± 8.05 19.7 ± 2.9* 36.4 ± 4.5* 48.99± 5.14^(#@) 69.4 ± 17.7^(#@) cell number, 10⁶/L SRBC agglutinin titer(log₂) 5 6.03 ± 0.71 1.05 ± 0.28* 1.62 ± 0.27* 4.08 ± 0.58*^(#) 5.13 ±0.53*^(#) Differences are statistically significant (p < 0.05) ascompared: *—to the group of intact animals; ^(#)—to the control group(CP + normal saline); ^(@)—to the group of animals treated with GSH.

TABLE 7 Effect of GSSG in combination with 0.1% cystamine on IL-2 andGM-CSF production by splenocytes, bone marrow and blood cellularindices, and immune response to SRBC in cyclophosphamide treated mice.(M ± m) Intact animals Cyclophosphamide-treated animals Normal NormalGSSG + Parameter n saline saline GSH GSSG 0.1% inosine IL-2 productionby 10 43.5 ± 4.01 14.0 ± 2.7* 20.3 ± 2.6* 30.9 ± 3.03^(#@) 38.8 ±4.53^(#@) splenocytes, (U/mL) GM-CSF production by 10 190.5 ± 18.4 42.0± 5.7* 66.7 ± 7.5* 137.0 ± 13.09^(#@) 183.7 ± 17.8^(#@) splenocytes,(colonies/10⁵ cells) Blood leukocyte count, 10 12.3 ± 1.28 4.95 ± 0.88*6.2 ± 1.06* 7.8 ± 0.84^(#@) 10.5 ± 1.56^(#@) 10⁹/L Blood lymphocytecount, 10 8.2 ± 0.72 3.6 ± 0.63* 5.31 ± 0.77* 7.2 ± 0.96^(#) 7.8 ±0.84^(#) 10⁹/L Bone marrow nucleated 10 61.3 ± 5.9 28.5 ± 4.2* 36.4 ±4.5* 48.9 ± 5.14^(#@) 56.7 ± 4.91^(#@) cell nubmer, 10⁶/L SRBCagglutinin titer (log₂)  5 6.03 ± 0.60 1.78 ± 0.36* 2.09 ± 0.37* 4.08 ±0.57*^(#) 4.29 ± 0.41*^(#) Differences are statistically significant (p< 0.05) as compared: *—to the group of intact naimals; ^(#)—to thecontrol group (CP + normal saline); ^(@)—to the group of animals treatedwith GSH.

EXAMPLE #3 Effect of GSSG and its Drug Forms on Cytokine and HemopoieticFactor Production as Well as on Hemopoiesis and Immunity Parameters inRadiation-induced Hemo- and Immunodepression

Both oxidized (GSSG) and reduced (GSH) glutathione, as well as GSSG drugforms containing 0.003% hydrogen peroxide, or 0.1% inosine, or 0.1%cystamine, were evaluated in a murine model of hemo- andimmunodepression induced by a single irradiation in a total dose of 1Gy.

The study was designed to evaluate efficacy of 7-day dailyadministration of the test articles (with the dosing started 2 hourspost-exposure) on the capability of the splenocytes from mice exposed toradiation to produce IL-2 and GM-CSF in vitro. In addition, the numberof blood leukocytes and lymphocytes and the spleen and bone marrowcellularity (number of karyocytes), as well as splenic and medullarycolony-stimulating capacity, were determined at 8 days post-exposure.

Male CBA mice (18 to 20 g body weight) were irradiated with single doseof 180 kV X-rays filtered with 0.5 mm Cu (at 15 mA, distance—70 cm,duration 2 min. and 28 sec.).

The total absorbed dose comprised approximately 1 Gy. Five groups ofanimals (with no less than 12 mice in each) were formed. Groupdescription is represented below.

Control Groups:

#1—intact animals receiving a sham irradiation procedure to reproduce astress impact, which further were treated with test article vehicle(normal saline);

#2—control animals irradiated in a dose of 1 Gy, which further weretreated with test article vehicle (normal saline);

#3—animals irradiated in a dose of Gy, which further were treated with sreference article (GSH dissolved in normal saline) in a dose of 5 mg/kg;

Test Groups:

#4—animals irradiated in a dose of Gy, which further were treated withthe test article (GSSG dissolved in normal saline) in a dose of 5 mg/kg;

#5—animals irradiated in a dose of Gy, which further were treated with avariant of the test article drugform (GSSG dissolved in normal salinecontaining 0.003% H₂O₂) with a GSSG dose of 5 mg/kg;

#6—animals irradiated in a dose of Gy, which further were treated withGSSG in normal saline containing 0.1% inosine) with a GSSG dose of 5mg/kg;

#7—animals irradiated in a dose of 1 Gy, which further were treated withGSSG in normal saline containing 0.1% cystamine) with a GSSG dose of 5mg/kg;

Two hours after the irradiation the intraperitoneal injections of thetest or reference articles were started (as it has been describedabove). Injections were performed during 7 days: once a day, daily.

Twenty four hours after the completion of 7 day treatment course (on the8th day after the irradiation). mice were euthanized and splenocytecultures were aseptically prepared for assessment of spontaneousproduction of IL-2 and GM-CSF by the spleen lymphocytes in vitro.

Simultaneously, blood, spleen and marrow samples were collected forblood leukocyte and lymphocyte, and spleen and marrow nucleated cellcounting.

Additionally, hemopoietic colony formation ability of spleen and bonemarrow cells was, assessed by the method of direct count of colonyforming units (CFU) in the spleens of irradiated CBA mice receivingintravenously spleen or bone marrow cells obtained from animals ofcontrol or test groups.

Splenocytic IL-2 and GM-CSF levels, blood, bone marrow, and spleencellular indices as well as colony-stimulating capacity numbers(colony-forming units, CFU) in the bone marrow and spleen of theirradiated animals at 8 days post-exposure, are summarized in Tables 8,9, 10.

TABLE 8 Effect of the test articles on IL-2 and GM-CSF production bysplenocytes, bone marrow, spleen and blood cellular indices, and bonemarrow and spleen hematopoietic colony formation capability inirradiated mice. (M ± m) Sham irradiated animals Irradiated animalsNormal Normal GSSGO + Parameter n saline saline GSH GSSG H₂O₂ IL-2production 12 41.2 ± 4.4 5.0 ± 0.5* 8.6 ± 1.3* 25.1 ± 4.9*^(#@) 37.1 ±3.4^(#@) by splenocytes, (U/mL) GM-CSF 12 120.2 ± 12.4 20.7 ± 8.6* 31.8± 3.9* 93.1 ± 11.5^(#@) 106.4 ± 5.2^(#@) production by splenocytes,(colonies/10⁵ cells) Blood leukocyte 12 12.7 ± 1.3 3.4 ± 0.9* 4.8 ± 0.8*8.7 ± 1.3*^(#@) 10.7 ± 2.0^(#@) count, 10⁹/L Blood 12 7.9 ± 0.7 2.2 ±1.3* 3.4 ± 0.6* 5.9 ± 0.8^(#@) 6.9 ± 0.8^(#@) lymphocyte count, 10⁹/LSpleen nucleated 12 9.8 ± 1.5 4.8 ± 1.3* 4.3 ± 1.5* 7.7 ± 1.2^(#@) 8.2 ±2.0^(#@) cell number, 10⁷/L Bone marrow 12 4.51 ± 3.2 14.0 ± 1.0* 17.2 ±3.5* 33.3 ± 4.2*^(#@) 37.0 ± 4.0*^(@) nucleated cell number, 10⁶/L Bonemarrow 12 59.4 ± 3.2 11.6 ± 2.2* 22.1 ± 3.6* 44.3 ± 3.9*^(#@) 49.3 ±3.9^(#@) CFU Spleen CFU 12 93.2 ± 4.1 40.0 ± 5.4* 56.3 ± 6.8* 88.3 ±6.8^(#@) 87.6 ± 4.7^(#@) Differences are statistically significant (p <0.05) as compared: *—to the group of intact naimals; ^(#)—to the controlgroup (CP + normal saline); ^(@)—to the group of animals treated withGSH.

TABLE 9 Effect of GSSG in combination with 0.1% cystamine on IL-2 andGM-CSF production by splenocytes, bone marrow, spleen and blood cellularindices, and bone marrow and spleen hematopoietic colony formationcapability in irradiated mice. (M ± m) Sham- irradiated Irradiatedanimals animals GSSG + 0.1% Parameter n Normal saline Normal saline GSHGSSG cystamine IL-2 production by 12 45.4 ± 4.2 5.6 ± 0.71* 9.3 ± 1.44*29.3 ± 3.18*^(#@) 40.1 ± 4.10^(#@) splenocytes, (U/mL) GM-CSF productionby 12 132 ± 11.8 28.6 ± 4.5* 34.3 ± 3.99* 103 ± 11.6^(#@) 113 ±9.07^(#@) splenocytes, (colonies/10⁵ cells) Blood leukocyte count, 1213.3 ± 1.08 3.1 ± 0.9* 5.7 ± 0.9* 9.3 ± 4.5*^(#@) 11.2 ± 1.83^(#@) 10⁹/LBlood lymphocyte count, 12 8.6 ± 0.74 3.38 ± 0.61* 4.6 ± 0.70* 6.79 ±0.82^(#@) 7.12 ± 0.899^(#@) 10⁹/L Spleen nucleated cell 12 10.5 ± 0.975.8 ± 0.9* 6.93 ± 0.85* 8.9 ± 1.07^(#@) 10.7 ± 1.13^(#@) number, 10⁷/LBone marrow nucleated 12 48.3 ± 3.8 15.1 ± 1.69* 24.7 ± 3.0* 39.5 ±4.17*^(#@) 5.10 ± 4.81^(#@) cell number, 10⁶/L Bone marrow CFU 12 61.3 ±5.2 16.0 ± 2.5* 25.6 ± 3.99* 50.3 ± 5.14*^(#@) 55.7 ± 5.31^(#@) SpleenCFU 12 104 ± 9.2 43.5 ± 5.8* 66.3 ± 7.07* 94.0 ± 8.81^(#@) 107 ±11.7^(#@) Differences are statistically significant (p < 0.05) ascompared: *—to the group of intact animals; ^(#)—to the control group(CP + normal saline); ^(@)—to the group of animals treated with GSH.

TABLE 10 Effect of GSSG in combination with 0.1% inosine on IL-2 andGM-CSF production by splenocytes, bone marrow, spleen and blood cellularindices, and bone marrow and spleen hematopoietic colony formationcapability in irradiated mice. (M ± m) Sham- irradiated Irradiatedanimals animals GSSG + 0.1% Parameter n Normal saline Normal saline GSHGSSG inosine IL-2 production by 12 45.1 ± 4.3 4.6 ± 0.53* 9.9 ± 1.08*26.9 ± 3.4*^(#@) 44.3 ± 4.71^(#@) splenocytes, (U/mL) GM-CSF productionby 12 132 ± 11.9 21.8 ± 3.7* 35.9 ± 4.15* 116 ± 11.7^(#@) 163 ±22.1^(#@) splenocytes. (colonies/10⁵ cells) Blood leukocyte count, 1212.0 ± 1.4 3.04 ± 0.81* 4.95 ± 0.62* 7.93 ± 0.96*^(#@) 10.9 ± 2.04^(#@)10⁹/L Blood lymphocyte count, 12 8.15 ± 0.76 1.94 ± 0.51* 4.0 ± 0.58*6.7 ± 0.83^(#@) 7.8 ± 0.86^(#@) 10⁹/L Spleen nucleated cell 12 9.91 ±1.3 3.5 ± 0.66* 5.5 ± 0.70* 9.0 ± 1.13^(#@) 10.2 ± 1.5^(#@) number,10⁷/L Bone marrow nucleated 12 47.3 ± 3.18 13.0 ± 1.8* 22.5 ± 3.08* 39.9± 4.5*^(#@) 51.7 ± 4.98^(#@) cell number, 10⁶/L Bone marrow CFU 12 56.2± 4.4 9.7 ± 1.3* 25.3 ± 3.7* 48.9 ± 5.13*^(#@) 69.0 ± 7.08^(#@) SpleenCFU 12 154 ± 9.45 35.0 ± 5.14* 59.8 ± 6.18* 99.3 ± 10.11^(#@) 167.0 ±17.3^(#@) Differences are statistically significant (p < 0.05) ascompared: *—to the group of intact animals; ^(#)—to the control group(CP + normal saline); ^(@)—to the group of animals treated with GSH.

As is evident from the data of the tables, administration of GSSG, orits drug forms containing 0.003% hydrogen peroxide, or 0.1% inosine, or0.1% cystamine, results in statistically significant recovery of IL-2and GM-CSF production by splenocytes, whereas GSH produces nosignificant effect.

Furthermore, both GSSG alone and its pharmacologically activecompositions exerted a significant normalizing effect on the blood,spleen, and bone marrow cellularity. In several instances the effect ofGSSG dissolved in hydrogen peroxide has been found to be more prominent.For example, while GSSGper se exhibited no statistically significanteffect (as compared to controls) on IL-2 splenocytic production, bloodleukocytes, bone marrow cellularity, and bone marrow colonies, GSSG inhydrogen peroxide did produce a statistically meaningful effect. Ifcompared with hydrogen peroxide, both inosine and cystamine were foundto exert more prominent effect of enhancing the GSSG action, with themaximal effect being achieved in case of active composition of GSSG withinosine.

Thus, the use of the subject method in animals developedradiation-induced hemo- and immunodepression results in pronouncedstimulation of the endogenous IL-2 and GM-CSF production, and also leadsto an accelerated recovery of the cellular compositions of the blood,lymphoid and hemopoietic organs as well as colony-forming activity ofthe bone marrow and spleen.

EXAMPLE #4 Effect of GSSG and its Drug Forms on the Process ofProliferation and Apoptosis of Normal and Tumor Cells

The ability of oxidized glutathione (GSSG), as well as its drug formscontaining 0.003% hydrogen peroxide, or 0.1% inosine or 0.1% cystamine,to influence processes of a cellular proliferation and/or death wasevaluated using normal or tumor cells. To this end, GSSG, or its drugforms had been incubated for 24 hours with cells of myeloid line HL-60and normal human lymphocytes isolated form peripheral blood of healthyvolunteers. Subsequent evaluation of the cell cycle parameters wascarried out by the flow cytofluorometry technique.

Venous blood of a healthy volunteer was collected into heparinizedtest-tubes which had been tested for endotoxin. A mononuclear fractionof blood leukocytes were obtained by centrifugation in gradient offikoll-metrizoat (Histopaque, Sigma). Cell concentration was adjusted to2×10⁶ cells per 1 ml of “complete” cell culture medium (RPMI 1640),containing 20 mM HEPES, 2 mM glutamine, 50 μg/mL gentamicin and 10%fetal calf serum. Cell viability was estimated by the Trypan blueexclusion method, then the cell suspension was placed into wells of96-well microtiter plates—200,000 cells per well. Cells of HL-60 linewere grown in RPMI-1640 medium with the addition of 10% fetal calfserum. Cultivation was carried out in closed flasks, the medium volumewas 12 mL, it was changed every four days by centrifugation. The natureof the cells growth was suspensive. Evaluation of the test solution ofGSSG (5000 μg/mL), as well as GSSG solutions containing 0.003% hydrogenperoxide, or 0.1% cystamine, was carried out using 6 cellular samples ofnormnal lymphocytes and HL-60 cells for each test solution. 50 μL ofeach test solution were added to one or the other cell culture andthereafter cells were cultivated for 24-96 hours. Then, they were testedby the flow cytofluorometry to estimate DNA content in the cell nuclei.In case of apoptosis-like cellular death, the portion of cell nucleiwith normal content of DNA became reduced, while the portion of cellnuclei containing abnormally small DNA quantity became larger.

The analysis procedure was the following: after incubation completion,cells were centrifuged and transferred to a standard phosphate isotonicbuffer pH 7.4, containing RNA-ase A (20 μg/mL), ethidium bromide(fluorometric indicator for double stranded nucleic acid. 10 μg/mL) andMgCl₂ (5 mM). After the cells were disintegrated by nonionic detergentTriton X-100 (final concentration 0.1%). The suspension of cell nucleithus obtained was analyzed by flow cytofluorometry with an argon laseras a source of light (wave length 488 nm). The red fluorescence due toDNA bound ethidium bromide was taken to be the measure of DNA content inthe cell nuclei. In addition, corresponding samples were studiedmicroscopically for revealing concomitant changes in cell morphology.

The study results are presented in Tables 11, 12 and FIG. 1). The table11 shows the presence of GSSG or its drug forms promoted proliferationof normal lymphocytes of healthy. volunteers, which resulted in increasein their number, while flow cytofluorometry analysis did not reveal anychanges characteristic for apoptosis-like cell death (FIG. 1c-d).

Observation carried out on cell cultures of the tumor cells of myeloidline HL-60 revealed ability of GSSG (as well as its drug forms) toslowdown the proliferation of transformed cells. Table 12 shows thatGSSG compositions with hydrogen peroxide, inosine and cystamine inhibitcell HL-60 proliferation better than GSSG alone. The flowcytofluorometry analysis demonstrates the slowdown of cell growth of theHL-60 line cells was associated with characteristic morphologicalindications of apoptosis-like death: sphere-like cells becamemulti-fragmented with plural interceptions, the number of cell nucleiwith normal content of DNA fell down, while there was an increase inportion of nuclei with abnormally low DNA content (FIGS. 1a-1 b).

TABLE 11 Effect of the test articles on number of normal lymphocytes perwell (× 10⁴ cells) throughout the 96-hr incubation. (M ± m) Testarticles (solutions) 24 hours 48 hours 72 hours 96 hours GSSG in normalsaline 27 ± 2  98 ± 6* 176 ± 12 386 ± 18* GSSG + 0.003% H₂O₂ 25 ± 4 108± 8* 231 ± 14* 419 ± 21* GSSG + 0.1% inosine 28 ± 3 107 ± 5* 212 ± 16*306 ± 12* GSSG + 0.1% cystamine 26 ± 3  93 ± 5* 186 ± 10* 263 ± 14*0.003% H₂O₂ 28 ± 2  73 ± 5 123 ± 8 206 ± 8 0.1% inosine 26 ± 4  78 ± 7141 ± 12 216 ± 16 0.1% cystamine 30 ± 2  72 ± 4 122 ± 9 196 ± 11 10%fetal calf serum 29 ± 4  74 ± 7 133 ± 18 263 ± 13 *Differences arestatistically significant (p < 0.05) as compared to 10% fetal calfserum.

TABLE 12 Effect of the test articles on number of HL-60 cells per well(× 10⁴ cells) throughout the 96-hr incubation. (M ± m) Test articles(solutions) 24 hours 48 hours 72 hours 96 hours GSSG in normal saline102 ± 4 156 ± 6* 386 ± 21* 390 ± 11* GSSG + 0.003% in H₂O₃  96 ± 6* 132± 4* 286 ± 18* 306 ± 18* GSSG + 0.1% inosine  49 ± 3*  76 ± 6* 138 ± 11*165 ± 9* GSSG + 0.1% cystamine  68 ± 8* 102 ± 11* 242 ± 19* 256 ± 14*0.003% H₂O₂ 122 ± 6 186 ± 12 488 ± 24 712 ± 22 0.1% inosine  96 ± 8* 152± 8* 312 ± 21* 527 ± 18* 0.1% cystamine 112 ± 10 182 ± 9 465 ± 11 618 ±19 10% fetal calf serum 119 ± 7 181 ± 13 471 ± 7 752 ± 16 *Differencesare statistically significant (p < 0.05) as compared to 10% fetal calfserum.

Thus, the results obtained enable to declare the dual functionalproperties of GSSG and its drug forms which selectively induceproliferation slowdown and apoptosis-like death of tumor cells whileaccelerate proliferation of normal human cells (lymphocytes) without anysigns of their apoptosis. The application of GSSG in combination withinosine produces the most prominent effect of GSSG in respect of normalcells.

EXAMPLE #5 Effect of GSSG and its Drug Forms on Progression ofExperimental Tumors in Mice

An antitumor activity of GSSG, as well as its drug forms containing0.003% hydrogen peroxide or 0.1% inosine, or 0.1% cystamine, wasevaluated in the two mouse models of the tumor process induced by theintraperitoneal inoculation of leukemia P388 and leukemia L1210 cells.An influence of 7 day course of test article daily administration wasstudied in respect of variations of serum cytokine levels (IL-1, IL-2,IL-6, IFNα, TNF). In parallel, the tumor progression was estimated usingthe two integral indices: pace of mouse weight gain due to accumulationof ascitic fluid, and by animal mean survival time after inoculation.

The study was carried out on DBA/2 mice weighing 18-21 g. First, tumorcell passage was performed using 6 animals for each cell line. For this,cells kept at the temperature of the liquid nitrogen were de-frozen andadjusted to the concentration of 5×10⁶ cells/mL by sterile Hanks'solution. Then, 6 mice were intraperitoneally inoculated with 0.2 mL ofeach line cellular suspension.

Ascitic fluid was collected 6 days after the inoculation with L1210cells and 8 days after the inoculation with P388 ones. Thus obtained,the samples of passaged tumor cells were used for the main experiments.The fluid liquid was dissolved by sterile Hanks' solution so that cellconcentration be 5×10⁶ cells/mL for P388 cells and 5×10⁵ cells/mL forL1210 cells.

Nine groups of animals with no less than 15 mice each were formed forexperiments with either tumor cell line. Mice were inoculated with 0.2mL of resultant cell suspensions per mouse (10⁶ P388 cells/mouse, and10⁵ L1210 cells/mouse). 24 hours after the tumor cells inoculation.animals were given the first injections of the test articles orvehicles. The test article injections were made daily till the 14th dayof the experiment or until the animal's death. The volume of solutionsinjected comprised 0.01 mL/g body weight. Description of nine groups ofanimals formed for experiments with either tumor cell line is givenbelow.

Control Groups:

#1—intact animals receiving imitation of tumor cell inoculation(injection of normal saline) which further were treated with normalsaline throughout the entire experiment;

#2—control animals, inoculated with tumor cells, which further weretreated with test article vehicle (normal saline);

Control Groups:

#3—experimental animals, inoculated with tumor cells, which further weretreated with test article (GSSG dissolved in normal saline) in a dose of5 mg/kg;

#4—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (GSSG dissolved innormal saline containing 0.003% of hydrogen peroxide), with a GSSG doseof 5 mg/kg;

#5—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (GSSG dissolved innormal saline containing 0.1% of inosine), with a GSSG dose of 5 mg/kg:

#6—experimental animals. inoculated with tumor cells, which further weretreated with a variant of test article drug form (GSSG dissolved innormal saline containing 0.1% cystarnine), with a GSSG dose of 5 mg/kg:

#7—experimental animals, inoculated with tumor cells, which further weretreated with a variant of drug form component (normal saline containing0.03% of hydrogen peroxide), without GSSG;

#8—experimental animals, inoculated with tumor cells, which further weretreated with a variant of drug form component (normal saline containing0.1% of inosine), without GSSG;

#9—experimental animals, inoculated with tumor cells, which further weretreated with a variant of drug form component (normal saline containing0.1% of cystamine), without GSSG;

Tables 13 and 14 contain results on test article efficacy evaluation asto variations of cytokine endogenous production, as well as data onintegral parameters of the tumor process progression. The resultsobtained show that both GSSG and its drug forms have a substantialcytokine inducing effect, reliably retard (if compared to the controlgroups) the accumulation of ascitic fluid and increase the mean survivaltime. GSSG alone and GSSG together with 0.003% of hydrogen peroxideincrease more remarkably the IL-1 and IFNα serum levels, whereas GSSG incombination with 0.1% inosine and 0.1% cystamine cause a larger increasein IL-2, IL-6, TNFα serum levels.

The most prominent antitumor effect in respect to slowdown of asciticfluid accumulation and prolongation of the mean survival time for eithertumor models (P388 and L1210 leukemia) were obtained with GSSG incombination with 0.1% cystamine.

TABLE 13 Effect of the test articles on the cytokine serum levels, theaccumulation of ascitic fluid and the mean survival time of miceinoculated with leukemia L1210 cells (M ± m) Accumulation of asciticfluid The number Concentration of factors in serum, (pg/mL); (weightgain, Mean Group of animals of injections IL-1 IL-2 IL-6 IFNα TNFα %)survival time 1 2 3 4 5 6 7 8 9 Control animals 0 22.0 ± 14.50 ± 93.20 ±82.2 ± 79.70 ± 0.7 ± 9.02 ± 0.19 3.15 2.56 10.58 9.05 8.15 0.1 3 28.5 ±23.18 ± 108.0 ± 100.55 ± 80.3 ± 7.14 ± 4.01 3.11 14.12 11.34 8.81 0.9 713.4 ± 17.8 ± 136.70* ± 140.3 ± 196.90 ± 25.4 ± 2.68 2.51 15.2 16.2521.30 2.62 Intact animals 0 20.09 ± 13.14 ± 84.0 ± 108.0 ± 77.90 ± 0.2 ±35 ± 0  1.95 1.12 9.65 11.33 6.85 0.1 3 25.10 ± 21.75 ± 85.60 ± 101.0 ±89.0 ± 1.12 ± 2.31 1.44 9.01 8.72 7.13 0.3 7 21.30 ± 21.15 ± 84.9 ± 90.0± 116. ± 4.6 ± 2.98 1.86 7.16 10.11 10.83 1.23 GSSG 0 27.5 ± 14.7 ±124.40 ± 144.80 ± 98.10 ± 0.77 ± 10.74 ± 0.51* 3.60 3.13 13.7 15.3411.54 0.16 3 57.6 ± 57.7 ± 301.0 ± 408.0* ± 397.0* ± 4.02* ± 7.14 6.8032.2 54.3 44.50 0.53 7 167.5 ± 144.5 ± 678. ± 1207.0* ± 610.0* ± 15.67*± 18.30 17.03 74.5 116.3 71.9 1.70 GSSG + 0.003% H₂O₂ 0 19.8 ± 14.84 ±108.0 ± 119.40 ± 78.0 ± 0.44 ± 11.13 ± 0.49* 2.05 2.13 9.17 9.56 6.150.16 3 126.0 ± 99.0 ± 298. ± 238.0 ± 406.* ± 3.17* ± 13.9 11.3 24.5 18.935.3 0.41 7 123.5 ± 189.0 ± 445. ± 1413* ± 818* ± 14.04* ± 12.7 21.44.14 129. 73.5 1.1 GSSG + 0.1% inosine 0 25.5 ± 17.40 ± 104. ± 122.4 ±121.9 ± 0.63 ± 12.01 ± 0.49* 2.86 1.92 8.15 10.43 10.33 0.16 3 83.10 ±40.8 ± 512.* ± 628.* ± 565.* ± 1.75* ± 9.15 5.0 48.7 56.4 50.03 0.25 7238.0 ± 91.1 ± 106. ± 1650.* ± 1904.* ± 5.69* ± 29.56 11.08 9.14 148186.0 0.74 GSSC + 0.1% cystamine 0 23.14 ± 17.0 ± 102. ± 129.0 ± 101.5 ±0.76 ± 11.96 ± 0.59* 2.86 1.55 8.04 9.80 8.16 0.19 3 118.0 ± 59.16 ±145. ± 761* ± 357.0* ± 2.47* ± 13.42 7.55 11.8 59.4 28.30 0.28 7 189.20± 249. ± 400.0* ± 1700.* ± 709.0* ± 6.85* ± 21.0 22.7 32.5 163. 59.00.91 0.003% H₂O₂ 0 17.07 ± 16.18 ± 120.9 ± 133.7 ± 110. ± 0.79 ±  9.7 ±0.21 1.65 1.68 10.7 10.45 9.13 0.17 3 38.15 ± 23.5 ± 140. ± 189. ± 158.0± 6.12 ± 4.11 3.3 13.3 15.45 11.97 0.73 7 23.6 ± 45.5 ± 103. ± 209. ±220.0* ± 21.61 ± 3.05 5.8 9.18 18.30 24.5 2.55 0.1% inosine 0 41.0 ±17.80 ± 108. ± 117.3 ± 104.3 ± 0.61 ± 9.61 ± 0.18 4.23 1.49 9.03 10.819.17 0.14 3 55.6 ± 22.3 ± 91.0 ± 160.0 ± 130.0 ± 7.02 ± 6.17 2.14 8.812.47 10.85 0.64 7 36.40 ± 14.6 ± 119. ± 205. ± 157.0 ± 26.30 ± 4.811.53 10.5 21.3 15.80 2.57 0.1% cystamine 0 36.0 ± 16.9 ± 63.0 ± 115.0 ±88.6 ± 0.47 ± 9.53 ± 0.18 3.12 1.5 5.0 10.52 5.19 0.18 3 47.50 ± 17.30 ±70.0 ± 200. ± 185.0 ± 5.93 ± 5.17 1.46 12.6 18.0 16.70 0.47 7 28.0 ±22.8 ± 155.0 ± 137.0 ± 213.0 ± 21.17 ± 3.0 1.90 13.4 14.5 18.54 2.05Differences are statistically significant (p < 0.05) as compared ascompared to the control group

TABLE 14 Effect of the test articles on the cytokine serum levels, theaccumulation of ascitic fluid and the mean survival time of miceinoculated with leukemia P388 cells (M ± m) The Accumulation number ofascitic Mean of in- Concentration of factors in serum, (pg/mL); fluid(weight survival Group of animals jections IL-1 IL-2 IL-6 IFNα TNFαgain, %) time 1 2 3 4 5 6 7 8 9 Control animals 0 19.6 ± 3.85 10.5 ±1.59 86.18 ± 7.13  90.5 ± 7.76 85.0 ± 6.15  0.5 ± 0.07 9.6 ± 0.22 3 34.7± 5.42 26.7 ± 3.18 133.0 ± 15.2 113.0 ± 12.0 96.17 ± 8.2  6.9* ± 0.52 710.8 ± 2.34 20.3 ± 3.08 156.10* ± 20.0    158 ± 10.8  218* ± 22.03 28.2*± 2.9  Intact animals 0 25.12 ± 1.76  17.70 ± 1.84  104.50 ± 9.94  90.50± 7.19 88.64 ± 7.14  0.3 ± 0.2 35* ± 0   3 33.0 ± 3.57 26.8 ± 3.07 92.80± 8.03  116.0 ± 10.55 89.0 ± 7.23 1.62 ± 0.4  7 30.83 ± 2.15  25.40 ±2.17  102.0 ± 8.89 112.31 ± 10.86 93.7 ± 7.64  5.1 ± 1.08 GSSG 0 23.5 ±4.22 12.8 ± 1.95 102.0 ± 12.8 134. ± 9.8 90.03 ± 8.07   0.48 ± 0.03211.0 ± 0.44* 3 62.3 ± 9.15 64.6 ± 7.13 280.0* ± 31.2   460. ± 40.8 306.± 24.4 3.7* ± 0.32 7 147.0 ± 17.30 128.10 ± 16.55  624.0* ± 45.6  1024.± 97.0 560. ± 48.8 15.2* ± 0.16  GSSG + 0.003% 0 17.4 ± 2.4  9.41 ± 2.02 90.8 ± 10.10 101.0 ± 9.88 73.5 ± 5.17 0.39 ± 0.11 11.6 ± 0.53* H₂O₂ 3109.6 ± 14.4  104.8 ± 15.30 314.0 ± 37.2 255.0 ± 22.3 355.* ± 36.2 2.93* ± 0.33  7 142.0 ± 16.3  174.0 ± 20.9  501.0* ± 48.3    1505 ±131.0 890.* ± 78.3  13.6* ± 0.64  GSSG + 0.1% 0 28.7 ± 3.05 7.13 ± 0.98129.8 ± 14.0  123.4 ± 10.01 109.0 ± 11.2  0.56 ± 0.16 12.7 ± 0.51*inosine 3 75.0 ± 8.13 36.4 ± 4.8  618.0* ± 52.3  693.0* ± 61.8  517.* ±44.5  1.64* ± 0.19  7 210.4 ± 26.8   84.0 ± 10.03 520.0* ± 51.0  1810.*± 129.  2120.* ± 193.  5.15* ± 0.59  GSSC + 0.1% 0 20.8 ± 2.91 16.7 ±1.88 118.9 ± 12.3 114.6 ± 9.87 95.6 ± 9.1  0.61 ± 0.15 12.5 ± 0.56*cystamine 3 109.2 ± 10.45 37.03 ± 4.15  156.6 ± 11.8 708.0* ± 61.9  326* ± 28.7  2.26* ± 0.17  7 168.0 ± 21.15 211.0 ± 25.6  414.0* ± 18.4  1950* ± 180.0 785.* ± 69.0  6.08* ± 0.77  0.003% H₂O₂ 0 15.5 ± 2.0414.95 ± 2.16  134.0 ± 15.6  129. ± 10.0 119. ± 9.13 0.63 ± 0.15 9.9 ±0.24 3 44.7 ± 6.14 22.0 ± 2.81 156.0 ± 16.3 205.8 ± 18.3 144.5 ± 12.8  5.4 ± 0.62 7 28.6 ± 4.11 40.8 ± 5.12 110.9 ± 12.5  190. ± 16.7 248. ±20.7 20.3 ± 2.28 0.1% inosine 0 36.7 ± 5.12 16.50 ± 1.09  115.0 ± 12.5 81.4 ± 6.13 122.0 ± 10.0  0.58 ± 0.13 9.8 ± 0.21 3 48.2 ± 7.13 20.19 ±1.54   90.0 ± 7.11  105. ± 11.3 96.5 ± 8.7  6.8 ± 0.8 7 31.0 ± 5.1213.40 ± 1.68  129.0 ± 10.4  184. ± 16.1 144.8 ± 12.9  25.0 ± 2.22 0.1%cystamine 0 30.0 ± 4.02 14.9 ± 2.05  72.7 ± 9.10   107 ± 8.06 80.5 ±7.14 0.67 ± 0.22 9.93 ± 0.27  3 41.5 ± 5.81 15.25 ± 1.80  184.0 ± 15.6 216. ± 19.08 204. ± 16.1  6.0 ± 0.49 7 22.3 ± 3.0  20.18 ± 2.50  170.6± 14.3  315. ± 9.80 220. ± 19.1 19.9 ± 1.67 Differences arestatistically significant (p < 0.05) as compared as compared to thecontrol group

Therefore, animal treatment according to present invention led to:significant increasing in endogenous production of IL-2, IL-6, IFNα andTNFα; and a reliable inhibition of progression of experimental tumorsand prolongation of the mean survival time.

New properties of a previously known substance—oxidized glutathione(GSSG), and its pharmacologically active compositions, containing 0.003%hydrogen peroxide, or 0.1% of inosine, or 0.1% cystamine, found in thepre-clinical studies, are thought to be sufficient to declare that GSSGand its pharmacological formulations possess an obvious biological andpharmacological activity, as well as a therapeutic effect. Thisjustifies the application of the corresponding drug forms of GSSG alongand GSSG in combination with pharmaceutically acceptable componentscapable of extending the oxidized glutathione half life, for preventingand treating the diseases in which stimulation of endogenous productionof cytokines and hemopoietic factors is advantageous and consideredbeneficial by those who are skilled in the art.

The following examples (##6-12) of the GSSG drug forms clinical usesupport the idea of utilizing GSSG as an inducer of the endogenouscytokine and hemopoietic factor production in man. and provide for themethod for disease treatment based on the above GSSG properties.

EXAMPLE #6 Effect of GSSG Drug Form on the Endogenous and ErythropoietinProduction in Patients having Neoplastic Disease

Data presented in this example demonstrate the GSSG stimulatory effecton the endogenous cytokine and hemopoietic factor production in cancerpatients. GSSG solution (5 mg/mL) was administered intravenously,slowly, every other day a 5 mg per injection. The cytokine endogenousproduction was determined by their blood levels prior to the firstadministration (with blood collected 24 hours before dosing) and afterthe third and the seventh injections. The cytokine levels were assessedby immunoenzyme technique using commercially available kits (Medgenix,Belgium), and expressed as pg/mL of culture medium.

As seen from the data given in Table 15, a pronounced stimulation of theendogenous cytokine (IL-1β, IL-6, TNF-α, IFN-α) and erythropoietin wasnoted as soon as after three first injections of GSSG. After the seventhadministration (14 days of treatment) a manifold increase in thecytokines and erythropoietin blood levels was observed in the majorityof cases.

TABLE 15 Effect of GSSG administered intravenously on cytokine anderythropoietin serum levels in cancer patients Number Serum level, pg/mLof in- erythro- Patients jections IL-1β IL-6 TNFα INFα poietin Pulmonaryadenocar- 0 18.3 138.0 57.2 83.3 143.0 cinoma with pleural 3 96.7 156.0280.0 395.6 605.0 metastases 7 104.6 150.0 315.0 378.0 548.0 Stomachadenocarci- 0 12.0 93.5 27.0 4.6 21.6 noma with liver 3 28.1 228.0 215.033.6 53.5 metastases 7 31.7 204.0 147.0 34.0 47.1 Suprarenal corticocy-0 8.4 61.9 39.8 41.3 8.3 toma with liver, pul- 3 12.9 105.0 113.0 56.032.4 monary and peritoneal 7 17.3 167.0 103.9 61.5 28.6 metastases

EXAMPLE #7 Stimulation of the Endogenous and Erythropoietin Productionin a Patient Suffering from Colorectal Cancer Complicated withChemotherapy-induced Hemodepression

A 44-year old female patient was operated on for colorectal mass grownthrough the ovary and metastases in the mesenteric and a mental lymphnodes (T₄N₃M₁). Postoperatively, 5-fluorouracil chemotherapy wasconducted (total course dose 5.5 g) with resultant severe hemotoxicity.

One month after the chemotherapy the patient was reexamined, andultrasonography of the peritoneum and computed tomography of the liverrevealed an oval-shaped 13×10 mm solitary metastasis in the left liverlobe. Repeat blood counts showed incomplete recovery of the bloodindices (leukopenia, lymphopenia, anemia, and thrombocytopenia ofvarious severity were noted) rendering further chemotherapy impossible.

Laboratory parameters prior to the use of the oxidized glutathione drugform (5 mg of GSSG in 1 mL of 0.003% hydrogen peroxide) are listed inTable 16. The treatment according to the subject method was commencedwith GSSG given intravenously for seven days, 5 mg once daily. After a3-day interval, the treatment was resumed with 15 mg daily dose, IV, 10days. This course was followed by a 7-day recess after which the therapywas continued with GSSG being given every other day IM, 15 mg daily (atotal of 20 injections).

50 days following commencement of the treatment the patient wasreevaluated, and ultrasonography of the peritoneum and computedtomography of the liver showed a considerable shrinkage (more than 50%of the pretreatment size) of the solitary hepatic metastasis. Thepost-treatment immunological indices are given in Table 16.

As seen from the data, both red and white. blood cell counts havesignificantly improved, platelets almost completely recovered, ESRreduced, CD4+, CD8+, NK cell numbers increased. A considerablestimulation of the endogenous cytokine and erythropoietin production,with TNF (together with increased natural killers) being probablyresponsible for the regression of the hepatic metastasis. These changeswere accompanied by an improved general condition of the patient.

This clinical case indicates apparent therapeutic efficacy of thesubject method. The administered therapy resulted in significantstimulation of the endogenous cytokine and hemopoietic factorproduction, reduction in hepatic metastasis size, normalization ofimmunity parameters, and overall improvement in the patient's wellness.

TABLE 16 Effect of GSSG on blood indices cytokine and erythropoietinserum levels, and immunological parameters in patent with colorectalcancer and chemotherapy induced hemodepression Parameter Prior to thetreatment After the treatment completion Erythrocytes 29 × 10¹²/L 4.1 ×10¹²/L Hemoglobin 79 g/L 108 g/L Leukocytes 3.6 × 10⁹/L 5.4 × 10⁹/LLymphocytes 0.67 × 10⁹/L 1.57 × 10⁹/L Platelets 92 × 10⁹/L 208 × 10⁹/LESR 44 mm/hr 19 mm/hr CD4* 204 × 10⁶/L 609 × 10⁶/L CD8* 255 × 10⁹/L 661× 10⁵/L NK-cells 39 × 10⁶/L 109 × 10⁶/L IL-1β 203 pg/mL 815 pg/mL IL-6318 pg/mL 1014 pg/mL TNFα 117 pg/mL 937 pg/mL IFNγ 84 pg/mL 506 pg/mLErythropoietin 162 pg/mL 618 pg/mL

EXAMPLE #8 Stimulation of the Endogenous Cytokine Production in an AIDSPatient with Cryptococcal Meningitis

A 28-year old male was admitted with a previously confirmed diagnosis ofAIDS, stage 3/4C (WHO staging system) in moderately grave condition. Thepatient presented with paroxysmal headache, dizziness, and vomiting.Weight 47 kg, Karnofsky score 60, torpid, fevers up to 39° C., dyspneaat rest.

Neurological examination revealed nuchal rigidity and diminished knee,ankle, biceps and triceps reflexes. Cerebrospinal fluid culture waspositive for Cryptococcus neoformans which served the basis for makingthe diagnosis of cryptococcus meningoencephalitis, and the AIDS stagewas refined as 4C.

A vigorous infusion therapy was started. In addition to palliativetherapy the patient received a course of Fungizone (Amphotericin B) withno positive outcome. The neurologic symptomatology and the patient'sgeneral state continued to deteriorate. A low to moderate grade fever(37.5-38.5° C.) persisted.

By the time oxidized glutathione was started (5 mg/mL), the patient hada significant drop in CD4+ and CD8+ peripheral blood counts as well asanemia and overall lymphopenia (see Table 17).

The patient received the treatment to the subject method for 3 months (1mL of the GSSG solution per administration). During the first month oftreatment the patient was dosed every other day (first 10 daysintravenously, the rest of the month—intramuscularly); during the secondmonth the patient received the drug every three days (first 10 days IV,the rest of the month—subcutaneously).

By the middle of the first month therapy,. the patient's conditionimproved significantly with the neurologic sign alleviated and low-gradefever not exceeding 37.5° C. In the course of treatment, the patient'scerebrospinal fluid was mycologically examined twice (cytology,cultures, latex-agglutination test for cryptococcal antigen). Towardsthe end of the first month therapy the number of viable Cryptococcalneoformans organisms was found to be considerably reduced. By the end ofthe second month the cytological, culture, and immunologic tests showedcerebrospinal fluid to be free of the pathogen. Because of the drasticimprovement in the patient's state, during the third month the drug wasgiven once weekly IM.

The hematology/immunology findings upon the therapy completion are givenin Table 17. As evident from the table, the anemia signs have reducedand a significant increase in the number of lymphocytes and theirsubsets has taken place. These findings constitute AIDS restaging from4C to 4B.

Noteworthy is the sizable elevation of the cytokine blood levels, withIL-2, IL-6, and IFN-γ playing the key role in the host defense againstpathogenic fungi.

At discharge, the patient's condition was found satisfactory with bodyweigh being 60 kg (weight gain made up 21.7% of the admission), normalbody temperature, Karnofsky score of 90, and no neurological signs.

TABLE 17 Effect of GSSG on blood indices, cytokine and erythropoietinserum levels, and immunological parameters in patient with AIDS andcryptococcal meningitis Parameter Pre-treatment Post-treatmentErythrocytes 3.1 × 10¹²/L; 3.9 × 10¹²/L; Hemoglobin 84 g/L; 126 g/L;Leukocytes 6.3 × 10⁹/L; 5.1 × 10⁹/L; Lymphocytes 0.8 × 10⁹/L; 1.45 ×10⁹/L; CD4* 55 × 10⁶/L; 338.3 × 10⁶/L; CD8* 135 × 10⁶/L; 883 × 10⁶/L;IL-1β 18.9 pg/mL; 123.4 pg/mL; IL-2 0.32 IU/mL 3.7 IU/mL IL-6 16.0pg/mL; 272.0 pg/mL; IL-10 45.0 pg/mL; 608.0 pg/mL; IFNα 27.0 pg/mL.314.0 pg/mL. IFNγ 15.7 pg/mL 349.8 pg/mL

EXAMPLE #9 Stimulation of the Endogenous Cytokine Production andTherapeutic Effect in Patients with AIDS Complicated by Isosporiasis

A 38-year old male had been observed for 2 years with the diagnosis ofAIDS, stage 3C (WHO Staging System). During the preceding year,recurrent episodes of oral and esophageal candidiasis had been recordedas well as chronic intestinal isosporiasis manifested by poor appetite,nausea, frequent vomiting and watery stools containing blood and mucus.Repeatedly used clotrimoxazole (trimethoprim plus sulfamethoxazole,TMP-SMX) had produced unsteady remissions with rapid recurrence of thesymptomatology. During the last month prior to admission another relapseof isosporiasis had occurred. The treatment with clotrimoxazole,immodium (loperamide) had brought no relief. The patient's condition hadbeen gradually deteriorating: refractory fever 38° C. and above, 6-7loose bloody and mucous stools a day, vomiting, advancing weight loss(15% of the premorbid weight in one year). The patient had been admittedwith progressive worsening of his condition.

On admission, the patient presented with moderately grave condition,Karnofsky score of 50. fever 38.2° C., emaciation (body weight 42 kg),virtually total lack of subcutaneous fat, pallor of skin, the signs oforal and esophageal candidiasis. Stool examination revealed a largenumber of Isospora belli oocysts.

By the time the therapy according to the subject method was started, thepatient had lymphopenia, marked decline in CD4+ and CD8+ lymphocytes,hypoproteinemia (see Table 18).

The patient received the oxidized glutathione drug form (5 mg of GSSG in1 mL 0.003% hydrogen peroxide) for 2 months (1 mL of the GSSG solutionper administration). During the first month of treatment the patient wasdosed every other day (first 10 days intravenously, the rest of themonth—intramuscularly); during the second month the patient received thedrug every three days (first 10 days IV, the rest of themonth—subcutaneously).

The patient's condition began to noticeably improve after the first twoweeks of treatment. By the end of the first month therapy the patientmoved bowels no more than 1 or,2 times a day with stools beingblood-free; body temperature only occasionally exceeded 37° C. At theend of the second month stool reexamination showed feces to be negativefor Isospora belli. Because of the drastic improvement in the patient'sstate, during the third month the drug was given prophylactically onceweekly IM. No relapses of the disease were noted.

The findings of hematology/blood chemistry evaluations upon the therapycompletion are given in Table 18. As seen from the table,hypoproteinemia has reduced, the number of lymphocytes and their subsetsconsiderably increased with the resultant restaging of AIDS to 3B stageaccording to the WHO Staging System.

Noteworthy is the marked increase of the cytokine blood levels, withIL-2 and IFN-γ known to play an important part in the host defenseagainst protozoan infections.

As a result of the therapy administered the patient's condition improveddrastically. fatigue alleviated, appetite regained. The weight gaincomprised 30% of the admission value, Kamofsky score—90. On physicalexamination the patient's condition was rated as satisfactory. During1.5 month follow-up no diarrhea relapses were reported.

TABLE 18 Effect of GSSG on blood indices, cytokine and erythropoietinserum levels, and immunological parameters in patient with AIDS andisosporiasis Parameter Pre-treatment Post-treatment Erythrocytes 4.04 ×10¹²/L 4.75 × 10¹²/L Hemoglobin 108 g/L 129 g/L Leukocytes 5.4 × 10⁹/L6.0 × 10⁹/L Lymphocytes 0.9 × 10⁹/L 1.8 × 10⁹/L CD4* 125 × 10⁶/L 436.5 ×10⁶/L CD8* 270 × 10⁶/L 949.3 × 10⁶/L Total protein 46 g/L 78 g/L IL-1β27.8 pg/mL 202.4 pg/mL IL-2 0.51 IU/ml 12.9 IU/ml IL-6 13.5 pg/mL 348.0pg/mL IL-10 62.0 pg/mL 956.0 pg/mL IFNα 148.3pg/mL 860.0 pg/mL IFNγ 61.2pg/mL 698.8 pg/mL

EXAMPLE #10 Stimulation of the Endogenous Erythropoietin Production andTherapeutic Effect in Patient with Hypoplastic Anemia and Pancytopenia

A 37-year old male had been observed for about a year with anemia ofunknown origin the severity of which had been gradually building up. For10 months he had been troubled with fatigability, dizziness, frequentnasal bleedings, unusual susceptibility to respiratory infections, threeepisodes of pneumonia with one of them being croupous pneumonia. Duringthe year the patient had lost 10% of his usual weight. Repeatedoutpatient treatment with oral and intravenous iron preparation, folicacid, B vitamins, including B,i, had produced no effect. One admissionthe patient presented with moderately grave condition, dyspnea onmoderate exertion, bruises, and isolated petechial spots. Successivehematology analyses have revealed moderately severe to severe fairlyhypochromic (color index 0.7-0.9) normocytic anemia (1.5-2.5×10₁₂/L),anisocytosis and poikilocytosis, moderate leukopenia, andthrombocytopenia within 50-80×10⁹/L.

An aggressive infusion therapy with iron;preparations, folic acid,cyanocobalamin, vitamins, prednisone, and repeated erythrocytetransfusions resulted in only marginal relief.

Bone marrow differential (punch biopsy) revealed marked hypocellularitywith medullary cavities populated predominantly with fat cells. Bothmyeloid and erythroid lineages are significantly suppressed with theerythroid/myeloid ration noticeably diminished. Megakaryocytes are scantin number with relative increase in nondifferentiated cells, plasmacells, and blasts. Iron stores are enriched. Diagnosis: hypoplasticanemia of unknown origin, pancytopenia.

Complete blood count and erythropoietin levels by the time the oxidizedglutathione drug form (5 mg GSSC in 1 mL of 0.003% hydrogen peroxide)was started are given in Table 19. As is evident from the table, thelaboratory findings are consistent with those characteristic ofhypoplastic anemia with no typical increase of erythropoietin bloodlevel, however. Moreover, the erythropoietin level was found to beconsiderably below the lower normal limit (9.2 pg/mL with the referencerange 30-170 pg/mL corresponding to 3-17 mIU/mL).

The oxidized glutathione formulation therapy was started withintramuscular injections of 1 mg GSSG b.i.d. for three days. Further thedose was escalated up to 5 mg b.i.d. for 7 days. Blood counts have shownless severe anemia. From that point, the drug form was dosed at 10 mg IMonce daily for 10 days and then, the RBC counts steadily recovering, thetherapy was switched to IV administration of GSSG, once every three daysfor 30 days. Vitamins and iron preparations were given concomitantly peros.

The hematology findings and erythropoietin levels obtained 50 daysfollowing the subject treatment onset are listed in Table 19. As is easyto see from the data, both RBS and WBC counts significantly improved, asdid the platelet counts, ESR reduced, erythropoietin levels exceeded theupper normal limit. Clinically, fatigue, dizziness, and dyspneadisappeared. On examination, no petechial spots or bruises could befound with no nasal bleedings observed or reported. The weight gain madeup 5.5 kg (8% of the premorbid weight).

Bone marrow reexamination (punch biopsy upon therapy completion) foundthe myeloid tissue to occupy 60% of the medullary cavities witherythroid/myeloid ratio in the myeloid tissue isles exceeding the norm.There were normoblastoid hyperplasia signs with megaloblastoid cellsfound in normoblast congregations. Mast cells were encountered,megakaryocytes were present in abundance. Iron stores appeared to besomewhat enriched.

This clinical case indicates a clear therapeutic efficacy of the drugform. Due to the treatment administered the initially suppressedendogenous erythropoietin production received a potent boost. As aresult, the hematology parameters virtually recovered and the anemiaclinical signs resolved. The patient was discharged in satisfactorycondition.

TABLE 19 Effect of GSSG on blood indices, erythropoietin serum level inpatient with hypoplastic anemia and pancytopenia Parameter Pre-treatmentPost-treatment Erythrocytes 1.8 × 10¹²/L 4.3 × 10¹²/L Hemoglobin 43 g/L119 g/L Color index 0.72 0.83 Reticulocytes 0.22% 2.85% Leukocytes 4.2 ×10⁹/L 7.2 × 10⁹/L Lymphocytes 1.6 × 10⁹/L 3.1 × 10⁹/L Platelets 72 ×10⁹/L 219 × 10⁹/L ESR 46 mm/hr 15 mm/hr Erythropoietin 9.2 pg/mL 201.7pg/mL

EXAMPLE #11 Stimulation of Endogenous Cytokine Production and theTherapeutic Effect in a Patient with a Stomach Cancer, PeritonealMetastases, Ascites, Plenomegaly and Cholestatic Hepatitis

A 33-year old patient was diagnosed as having stomach neoplasm for morethan 2 years (adenocarcinoma of moderate differentiation degree). In1993, the patient was operated on for a malignant stomach ulcer; andnumerous dense lymph nodes were found in the porte hepatis which wereconsidered to be metastases.

In January 1994 the course of chemotherapy (5FU) was complicated by thesevere cholestasis and percutaneous drainage of the left and right liverducts was undertaken, that 6 month's later was followed by thecholedochoejunostomy with changeable transliver drains with Brown'sanastomosis.

In November 1995 the state of the patient worsened. According to theexaminations the patient experienced an active secondary hepatitis. Theliver was enlarged and painful and protruded from the costal arch up to5-6 sm. Blood chemistry indices proved to be persistently abnormal:bilirubin—40.0 due to indirect (up to 31.0); activity of aminotransferases—approximately 6 times higher than upper normal limit,hypoalbunemia was up to 26%; and there was also hypergammaglobulinemia;hypercholesterolemia was up to 10.2 μmol/l.

During fibrogastrocopy (November, 1995), a tumor of the stomach locatedin the middle area of the stomach body and extending about 8 cm wasconfirmed. The tumor was solid-like. Stomach walls were rigid. Histologyexamination defined the tumor as adenocarcinoma of moderate degreelaparotomy. Ascites were found with plural metastases all over theperitoneum. splenomegaly. The patient was identified as inoperable.

A decision was taken to apply GSSG drug form containing 0.1% inosine.The drug was injected parenterally (intramuscular and intravenous), andadditionally, the drug form was used via local injections around thetumor tissue with the help of endoscope. An average dose which was usedfor intramuscular and intravenous injections—0.1-0.5 mg/kg, and forlocal injections—up to 50 mg in situ. Parenteral injections of the drugwere applied every other day, b.i.d. (intravenous injections at themorning, and intramuscular ones—at the evening), during three weeks, andafter that—two times in a week, during four weeks. Two months after thebeginning of the treatment with the drug form usedfibrogastroduodenoscopy showed that esophagus was passable, mucousmembrane was pink, cardia rosette was partly closed. On an emptystomach, a moderate amount of foamy secretion was in the stomach, whichwas intensively colored with bile. The tumor extent was 5 cm. At thesame time, substantial improvement of hematology and blood chemistryindices were found.

Four month's later, the liver protruded 1 cm beyond the rib arch. Onpalpation the liver was not painful. Supersonic examination showed theappearance of fibrous tissue instead on the place of some areaspreviously affected with tumor tissue. Fibrogastroduodenoscopy performedin May, 1996, showed that the esophagus was partly closed. There waslight turbid liquid in the stomach, which contained saliva. Mucousmembrane was pink. The tumor was 3.6 cm in extent with the stomach wallsbeing elastic. Duodenum was passable.

By comparison with results of examination conducted before treatmentwith the use of the GSSG drug form mentioned (November, 1995) the tumorwas shrunk in its extent for 55%. Simultaneously there were significantbeneficial changes in hepatic tests, hematology and immunology indices.

Thus, the treatment according to the present invention resulted inpartial regress of neoplastic process with simultaneous obviousbeneficial changes in hematology. blood chemistry and immunologyparameters, and significant improvement of life quality.

EXAMPLE #12 Stimulation of Endogenous Cytokine Production and theTherapeutic Effect in a Patient With Skin Cancer (Merkel's cellcarcinoma), Local Lymph Node Metastases and Chemotherapy-hemo- andInduced Immunodepression

A male patient, 64 years old, has been under medical supervision sinceAugust, 1995, when a hyperemic painless mass appeared in the scapulararea, which had progressively grown in size. After a month's time, themass spread over the axillary space, kept increasing, and becamepainful. A fever appeared (38.9° C.). Histological and immunologicalexamination in October, 1995 made the diagnosis clear: neuroendocrinalform of skin cancer (Merkel's cell carcinoma) stage III.

In December, 1995 the patient was given a course of CMF chemotherapy(cyclophosphamide+methotrexate+fluorouracil) without appreciablecurative effect. At the same time an obvious hemopoiesis depression(leukocytes 2.4×10⁹/L) developed with simultaneous growth of cervicaland superclavicular lymph nodes associated with local skin hyperemia.

In January-February 1996 chemotherapy scheme was changed:cysplatine+cyclophosphamide (CP instead of CMF). The chemotherapybrought about the following complications—cytopenia(leukocytes—1.4×10⁹/1), cardiotoxicity in the form of ischemiadeterioration. After the 2nd course of chemotherapy a substantial tumorprogression was observed: necrosis in the left subaxillary area withfistula formnation; edema of the left arm; infiltrating growth into softtissues in the area of the left shoulder and the left subaxillarytissues: intoxication; persistent fever (38.8° C.). Because ofinefficacy of chemotherapy and the obvious progression of the process,it was decided to administer a course of GSSG drug form in combinationwith 0.1% cystamine, together with chemotherapy (CMF).

After 10 daily injections of the GSSG drug form used (intravenously andintramuscularly, the dose 0.1-0.5 mg/kg per an injection), it wasnoticed: the following changes in the patient's status was revealed:improved quality of life (good appetite, mobility); ulceration dryingout, abolition of suppurative discharge; fistula scarring, 30% tumorshrinkage; normal body temperature; limitation of hyperemic areas, theimprovement of hematology indices.

The 3rd and 4th courses of chemotherapy (CMF) were carried out togetherwith GSSG drug form (intravenous and intramuscular injections, b.i.d.,intravenous dose 0.5 mg/kg; and intramuscular dose 0.2 mg/kg).Parenteral administration of the preparation was 3 times in a week, withlocal injections in the two spots around the tumor through the endoscopeonce a week (up to 25 mg for each spot). The following results wereobtained: tumor process regression; good endurance of chemotherapy, thedisappearance of pain syndrome, constant improvement of life quality,restoration of immunity and hemopoiesis, increasing level of cytokinesand hemopoietic factors (see table 20).

In two months the treatment with the use of the present invention therewas a stable level of endogenous production of cytokines and hemopoieticfactors; the diminution of the left cervical and supraclavicular lymphnodes; the 70% shrinkage of tumor size in two dimensions; positiveshifts in immunology indices; lack of chemotherapy hemo-depression.

The clinical observation proves the clear curative effect of thetreatment according to the present invention: together with the obviousstimulation of endogenous production of cytokines and hemopoieticfactors there were a substantial decrease in tumor size, improvement oflife quality, and beneficial changes in hematology, blood chemistry andimmunology parameters.

TABLE 20 Effect of GSSG on blood and immunology indices and cytokinelevels in patient with skin cancer (Merkel's cell carcinoma), locallymph node metastases and chemotherapy-induced hemo- andimmunodepression. Prior 3 months after the to the treatment Parametertreatment beginning Erythrocytes, 10¹²/L 3.9 4.1 Hemoglobin, g/L 112 114Platelets, 10⁹/L 210 262 Leukocytes, 10⁹/L 24 7.2 Neutrophils (stab), %6 8 Neutrophils (segm.), % 79 60 Lymphocytes, % 8 24 Monocytes, % 4 7Eosinophils, % 3 1 ESR, mm/hr 43 13 Total protein, g/L 61 78α1-globulin, % 9.20 2.3 α2-globulin, % 12.32 8.2 β-globulin, % 13.0814.0 γ-globulin, % 21.69 18.8 A/G ratio 0.78 0.94 Urea, mmol/L 8.54 4.3Creatinin, mmol/L 0.123 0.095 Bilirubin, mcmol/L 4.6 4.1 Prothrombinindex, % 82 100 Glucose, mmol/L 5.5 4.3 SGOT, mmol/hr/L 0.48 0.32 SGPT,mmol/hr/L 0.43 0.21 Lymphocytes, 10⁶/L 192 1728 B-lymphocytes (CD20*)10⁶/L 60 234 CD4*-lymphocytes, 10⁶/L 84 604 CD8*-lymphocytes, 10⁶/L 13329 CD4*/CD8* 6.5 1.8 IL2-receptor bearing cells (CD25*), 10⁶/L 64 881HLA11-receptor bearing cells, 10⁶/L 36 498 NK-cells (CD16+), 10⁶/L 24624 IgA, g/L 4.9 5.2

In Examples 13-15, the following designations for chemically modifiedGSSG derivatives will be used:

S-thioethylamine-glutathione disulfide S-thioethylamine-GSSGbis-[DL-6,8,thiooctanil]•glutathione disulfide bis-lipoil-GSSG[b-alanyl-L-hystidil]•glutathione disulfide carnosil-GSSG[9-β-D-ribofuranosyladenil]•glutathione adenosil-GSSG disulfidebis-[L-2-amino-4- bis-methionil-GSSG [methylthio]butanoil]•glutathionedisulfide

EXAMPLE 13 Effect of Lithium salt of GSSG, S-thioethylamine-GSSG andtheir Drug Forms on the Process of Apoptosis of Normal and Tumor Cells

The ability of S-thioethylamine-GSSG and lithium salt of oxidizedglutathione (GSSG), as well as their drug forms containing 0.003%hydrogen peroxide, or 0.1% inbsine or 0.1% cystamine, or 7% dimethylsulfoxide (DMSO) to influence processes cell death and apoptosisregulation was evaluated using normal or tumor cells (in all cases wherelithium salts and sodium salts are used in the Examples of theapplication, 2 atoms are present attached to sites X₁ and X₄ of theGSSG). To this end, these substances had been incubated for 72 hourswith cells of rat embrional fibroblasts (REF) and the same cells,transformed with adenoviral E1A gene in complementation withras-oncogene (e-ras cell line). Subsequent evaluation of the cellularityof experimental samples was carried out by counting the quantity of cellper milliliter (for REF cell line) or clones in dish (for e-ras cellline).

Cells were cultivated on DMEM medium supplemented with 10% fetal calfserum and 50 mg/mL gentamicin. Cultivation was carried out in Petridishes.

REF cells was cultivated with initial density of 800 000 cell per mL,evaluation of the cellularity was performed at 0, 24, 48 and 72 hours ofincubation with the test articles.

E-ras cells were seeded at a cell density of 300 cells per 5 cm dish. 7days after growth of e-ras cells had been started the quantity of cloneswas evaluated and the test articles were added into the dishes.

Evaluation of the test solution of lithium GSSG salt andS-thioethylamine-GSSG (5000 mg/mL), as well as lithium GSSG salt andS-thioethylamine- GSSG solutions containing 0.003% hydrogen peroxide,or, 0.1% inosine or 0.1% cystamine, or 7% DMSO, was carried out using 6cellular samples for each test solution.

50 mcL of each test solution were added to one or the other cell cultureand thereafter cells were cultivated for 24-72 hours. For thetest-system for estimation of apoptosis regulation UV-induced cell deathwas triggered in a dose of 4 Dj. Test articles were added immediatelyafter irradiation. Then, the quantity of cell per milliliter (for REFcell line) or clones in dish (for e-ras cell line) were monitored every24 hours. For determination of DNA-fragmentation the electrophoresis inagarose gel was used at standard settings.

The study results are presented in Tables 21-24. The tables 21 and 22show that the presence of lithium salt of GSSG or S-thioethylamine-GSSGor their drug forms didn't promote apoptosis of normal cells (REF line).Observation carried out on cell cultures of the e-ras cells revealedability of lithium salt of GSSG or S-thioethylamine-GSSG (as well as oftheir drug forms) to induce a transformed cell death.

TABLE 21 Effect of the test articles on number of REF cells (× 10³cells) throughout the 72-hr incubation (M ± m) Tests article (solution)0 hours 24 hours 48 hours 72 hours Li-GSSG in normal 860 ± 25 1496 ± 422606 ± 46 5180 ± 124 saline Li-GSSG + 0.003% 830 ± 17 1326 ± 34 2695 ±72 5360 ± 186 H2O2 Li-GSSG +0.1% 826 ± 12 1340 ± 64 2641 ± 77 5063 ± 134inosine Li-GSSG + 0.1% 831 ± 24 1329 ± 41 2831 ± 53 5302 ± 221 cystamineLi-GSSG + 7% 800 ± 22 1463 ± 26 2820 ± 48 5206 ± 210 DMSOS-thioethylamine- 789 ± 21 1422 ± 14 2602 ± 43 5112 ± 168 GSSG in normal782 ± 14 1426 ± 24 2645 ± 32 5160 ± 134 saline S-thioethylamine- 824 ±22 1398 ± 17 2684 ± 28 5210 ± 156 GSSG + 0.003% 841 ± 18 1386 ± 14 2478± 31 5089 ± 123 H2O2 S-thioethylamine- 821 ± 13 1462 ± 15 2671 ± 32 5121± 68  GSSG + 0.1% inosine S-thioethylamine- 822 ± 11 1365 ± 14 2598 ± 635059 ± 34  GSSG + 0.1% 811 ± 10 1426 ± 24 2642 ± 25 5034 ± 128 cystamineS-thioethylamine- 822 ± 14 1523 ± 11 2486 ± 34 5048 ± 126 GSSG + 7% DMSO801 ± 12 1420 ± 17 2651 ± 36 5298 ± 39  824 ± 21 1486 ± 46  2645 ± 1285125 ± 246 0.003% H2O2 0.1% inosine 0.1% cystamine 7% DMSO 10% fetalcalf serum

TABLE 22 Effect of the test articles on number of clones of e-ras cellsthroughout the 72-hr incubation (M ± m) Tests article (solution) 0 hours24 hours 48 hours 72 hours Li-GSSG in normal 260 ± 25 196 ± 22 146 ± 16108 ± 12 saline Li-GSSG + 0.003% 250 ± 17 186 ± 14 125 ± 12 106 ± 16H2O2 Li-GSSG +0.1% 248 ± 11 201 ± 11 134 ± 12  98 ± 14 inosine Li-GSSG +0.1% 254 ± 15 182 ± 10 121 ± 14 102 ± 11 cystamine Li-GSSG + 7% 261 ± 12184 ± 8  102 ± 16 76 ± 8 DMSO S-thioethylamine- 286 ± 14 202 ± 14 156 ±10 112 ± 12 GSSG in normal 271 ± 16 208 ± 12 152 ± 11 121 ± 10 salineS-thioethylamine- 292 ± 13 212 ± 14 151 ± 14 118 ± 14 GSSG + 0.003% 288± 11 210 ± 12 146 ± 8  124 ± 8  H2O2 S-thioethylamine- 278 ± 14 221 ± 8 132 ± 10 102 ± 8  GSSG + 0.1% inosine S-thioethylamine- 288 ± 4  286 ±4  264 ± 3  234 ± 6  GSSG + 0.1% 292 ± 11 290 ± 8  269 ± 8  243 ± 8 cystamine S-thioethylamine- 276 ± 4  281 ± 4  271 ± 6  258 ± 3  GSSG +7% DMSO 268 ± 11 271 ± 2  268 ± 8  243 ± 6  272 ± 8  275 ± 3  258 ± 4 232 ± 4  0.003% H2O2 0.1% inosine 0.1% cystamine 7% DMSO 10% fetal calfserum

The tables 23 and 24 show that the presence of lithium salt of GSSG orS-thioethylamine-GSSG or their drug forms do not promote the apoptosisof normal cells (REF line) induced by UV-irradiation. Observationscarried out on e-ras cell cultures revealed ability of lithium salt ofGSSG or S-thioethylamine-GSSG (as well as their drug forms) topotentiate death of the transformed cells.

TABLE 23 Effect of the test articles on number of REF cells (× 10³cells) throughout the 72-hr incubation (M ± m) after UV-irradiation.Tests article (solution) 0 hours 24 hours 48 hours 72 hours Li-GSSG innormal 860 ± 25 496 ± 42 260 ± 46 190 ± 24 saline Li-GSSG + 0.003% 830 ±17 326 ± 34 269 ± 22 193 ± 18 H2O2 Li-GSSG +0.1% 826 ± 12 340 ± 64 241 ±17 163 ± 13 inosine Li-GSSG + 0.1% 831 ± 24 329 ± 41 281 ± 33 192 ± 21cystamine Li-GSSG + 7% 800 ± 22 463 ± 26 282 ± 18 186 ± 10 DMSOS-thioethylamine- 789 ± 21 422 ± 14 260 ± 23 212 ± 16 GSSG in normal 782± 14 426 ± 24 245 ± 22 2160 ± 34  saline S-thioethylamine- 824 ± 22 398± 17 264 ± 22 210 ± 16 GSSG + 0.003% 841 ± 18 386 ± 14 248 ± 11 189 ± 23H2O2 S-thioethylamine- 821 ± 13 462 ± 15 271 ± 22 212 ± 16 GSSG + 0.1%inosine S-thioethylamine- 822 ± 11 365 ± 14 258 ± 33 159 ± 24 GSSG +0.1% 811 ± 10 426 ± 24 262 ± 22 234 ± 28 cystamine S-thioethylamine- 822± 14 523 ± 11 246 ± 34 208 ± 26 GSSG + 7% DMSO 801 ± 12 420 ± 17 251 ±36 208 ± 39 824 ± 21 486 ± 46 265 ± 28 195 ± 46 0.003% H2O2 0.1% inosine0.1% cystamine 7% DMSO 10% fetal calf serum

TABLE 24 Effect of the test articles on number of clones of e-ras cellsthroughout the 72-hr incubation (M ± m) after UV-irradiation. Testsarticle (solution) 0 hours 24 hours 48 hours 72 hours Li-GSSG in normal261 ± 20 166 ± 12 116 ± 6 78 ± 4 saline Li-GSSG + 0.003% 250 ± 12 146 ±14 115 ± 5 76 ± 6 H2O2 Li-GSSG +0.1% 248 ± 11 141 ± 11 124 ± 8 68 ± 4inosine Li-GSSG + 0.1% 254 ± 15 142 ± 10 101 ± 4  70 ± 11 cystamineLi-GSSG + 7% 261 ± 12 124 ± 8   86 ± 6 56 ± 8 DMSO S-thioethylamine- 286± 14 182 ± 14 116 ± 8 82 ± 4 GSSG in normal 271 ± 16 168 ± 12 102 ± 7 71 ± 10 saline S-thioethylamine- 292 ± 13 162 ± 7  111 ± 4 76 ± 4GSSG + 0.003% 288 ± 11 152 ± 8  116 ± 8 72 ± 8 H2O2 S-thioethylamine-278 ± 14 134 ± 8   82 ± 10 50 ± 6 GSSG + 0.1% inosine S-thioethylamine-288 ± 4  186 ± 4  124 ± 3 84 ± 6 GSSG + 0.1% 292 ± 11 190 ± 8  129 ± 893 ± 8 cystamine S-thioethylamine- 276 ± 4  181 ± 4  111 ± 6 108 ± 3 GSSG + 7% DMSO 268 ± 11 171 ± 2  108 ± 8 103 ± 6  272 ± 8  175 ± 3  125± 4 93 ± 4 0.003% H2O2 0.1% inosine 0.1% cystamine 7% DMSO 10% fetalcalf serum

Thus, the results obtained enable to postulate the duality of functionalproperties of lithium salt of GSSG or S-thioethylamine-GSSG and theirdrug forms which selectively induce apoptosis-like death of tumor cellswithout any signs of apoptosis in normal cells. Besides, all the testarticles were able to decrease the apoptosis processes of normal cellsinduced by UV-irradiation and, were able to potentiate these processesin transformed cells. The application of S-thioethylamine-GSSG incombination with DMSO produced a more prominent effect than that oflithium salt of GSSG in respect of transformed cells.

EXAMPLE 14 Effect of Lithium salt of GSSG and S-thioethylamine-GSSG andtheir Drug Forms on Progression of Experimental Tumors in Mice.

An antitumor activity of lithium salt of GSSG and S-thioethylamine-GSSG,as well as their drug forms containing 0.003% hydrogen peroxide, 0.1%inosine, 0.1% cystamine, or 7% dimethyl sulfoxide (DMSO) was evaluatedIn the three mouse models of the tumor process induced by theintraperitoneal inoculation of leukemia P388, leukemia L1210 cells andErlich adenocarcinoma cells. An influence of 7 day course of testarticle daily administration was studied in respect of tumorprogression, which was estimated using the two integral indices: pace ofmouse weight gain due to accumulation of ascitic fluid, and by animalmean survival time after inoculation.

The study was carried out on DBA/2 mice weighing 18-21 g. First, tumorcell passage was performed using 6 animals for each cell line. For this,cells kept at the temperature of the liquid nitrogen were de-frozen andadjusted to the concentration of 5×10⁶ cells/mL by sterile Hanks'solution. Then, 6 mice were intra peritoneally inoculated with 0.2 mL ofeach line cellular suspension.

Ascitic fluid was collected 6 days after the inoculation with L1210cells, 8 days after the inoculation with P388 ones and 18 days afterinoculation with Erlich adenocarcinoma cells. Thus obtained, the samplesof passaged tumor cells were used for the main experiments. The fluidliquid was dissolved by sterile Hanks' solution so that cellconcentration be 5×10⁶ cells/mL for P388 cells and Erlich adenocarcinomacells, 5×10⁵ cells/mL for L1210 cells.

Eleven groups of animals with no less than 15 mice each were formed forexperiments with either tumor cell line. Mice were inoculated with 0.2mL of resultant cell suspensions per mouse (10⁶ P388 and Erlichadenocarcinoma cells/mouse, and 10⁵ L1210 cells/mouse). 24 hours afterthe tumor cells inoculation, animals were given the first injections ofthe test articles or vehicles. The test article injections were madedaily till the 14^(th) day of the experiment or till the animal death.The volume of solutions injected comprised 0.01 mL/g body weight.

Description of nine groups of animals formed for experiments with eithertumor cell line is given below.

Control Groups:

#1—intact animals receiving imitation of tumor cell inoculation(injection of normal saline) which further were treated with normalsaline throughout the entire experiment;

#2—control animals, inoculated with tumor cells, which further weretreated with test article vehicle (normal saline);

Experimental Groups:

#3—experimental animals, inoculated with tumor cells, which further weretreated with test article (S-thioethylamine-GSSG dissolved in normalsaline) in a dose of 5 mg/kg;

#4—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (S-thioethylamine-GSSGdissolved in normal saline containing 0.003% of hydrogen peroxide), witha dose of 5 mg/kg;

#5—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (S-thioethylamine-GSSGdissolved in normal saline containing 0.1% of inosine), with a dose of 5mg/kg;

#6—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (S-thioethylamine-GSSGdissolved in normal saline containing 0.1% cystamine), with a dose of 5mg/kg;

#7—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (S-thioethylamine-GSSGdissolved in normal saline containing 7% DMSO), with a dose of 5 mg/kg;

#8—experimental animals, inoculated with tumor cells, which further weretreated with test article (Li salt of GSSG dissolved in normal saline)in a dose of 5 mg/kg;

#9—experimental animals, inoculated with tumor cells, which further weretreated with a variant of test article drug form (Li salt of GSSGdissolved in normal saline containing 0.003% of hydrogen peroxide), witha dose of 5 mg/kg;

#10—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of test article drug form (Li salt of GSSGdissolved in normal saline containing 0.1% of inosine), with a dose of 5mg/kg;

#11—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of test article drug form (Li salt of GSSGdissolved in normal saline containing 0.1% cystamine), with a dose of 5mg/kg;

#12—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of test article drug form (Li salt of GSSGdissolved in normal saline containing 7% DMSO), with a dose of 5 mg/kg;

#13—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of drug form component (normal salinecontaining 0.003% of hydrogen peroxide), without GSSG;

#14—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of drug form component (normal salinecontaining 0.1% of inosine), without GSSG;

#15—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of drug form component (normal salinecontaining 0.1% of cystamine), without GSSG;

#16—experimental animals, inoculated with tumor cells, which furtherwere treated with a variant of drug form component (normal salinecontaining 7% of DMSO), without GSSG;

Tables 25-27 contain results on test article efficacy evaluation onintegral parameters of the tumor process progression.

TABLE 25 Effect of the test articles on the accumulation of asciticfluid and the mean survival time of mice inoculated with leukemia L1210cells (M ± m) Accumulation of ascitic The number of (weight Mean Groupof animals injection gain, %) survival time Control animals 0 0.7 ± 0.19.02 ± 0.19 3 7.14 ± 0.9 7 25.4 ± 2.6 Intact animals 0 0.2 ± 0.1 35 ± 03 1.12 ± 0.3 7 4 ± 192 S-thioethylamine- 0 0.77 ± 0.2 19.2 ± 0.8 GSSG +normal saline 3 3.1 ± 0.4 7 92 ± 1.2 S-thioethylamine- 0.56 ± 0.2 19.8 ±0.6 GSSG + 0.003% H₂O₂ 3 2.9 ± 0.4 7 8.6 ± 1.4 S-thioethylamine- 0 0.42± 0.3 21.2 ± 0.8 GSSG + 0.1% inosine 3 2.2 ± 0.6 7 7.8 ± 1.4S-thioethylamine- 0 0.51 ± 0.1 19.8 ± 1.8 GSSG + 0.1% 3 3.4 ± 0.4cystamine 7 8.6 ± 2.1 S-thioethylamine 0 0.42 ± 0.3 22.2 ± 1.2 GSSG + 7%DMSO 3 2.8 ± 1.0 7 6.6 ± 2.1 Li salt of GSSG + 0 1.27 ± 0.2 12.2 ± 0.1normal saline 3 4.1 ± 0.4 7 12.2 ± 1.2 Li salt of GSSG + 0 1.56 ± 0.214.8 ± 0.6 0.003% H₂O₂ 3 4.9 ± 0.4 7 10.6 ± 1.7 Li salt of GSSG + 0 1.42± 0.3 12.2 ± 1.2 0.1% inosine 3 4.2 ± 0.6 7 9.8 ± 1.0 Li salt of GSSG +0 1.51 ± 0.1 13.8 ± 1.6 0.1% cystamine 3 3.4 ± 0.4 7 10.6 ± 1.7 Li saltof GSSG + 7% 0 2.42 ± 0.3 15.2 ± 1.4 DMSO 3 5.8 ± 1.0 7 12.6 ± 1.70.003% H₂O₂ 0 0.77 ± 0.2 9.2 ± 0.8 3 6.1 ± 0.4 7 19.2 ± 2.2 0.1% inosine0 0.56 ± 0.2 9.8 ± 0.6 3 7.9 ± 0.4 7 26.6 ± 2.4 0.1% cystamine 0 0.42 ±0.3 10.2 ± 0.8 3 8.2 ± 0.6 7 24.8 ± 2.1 7% DMSO 0 0.51 ± 0.1 10.2 ± 0.83 6.4 ± 1.4 7 23.6 ± 2.6

TABLE 26 Effect of the test articles on the accumulation of asciticfluid and the mean survival time of mice inoculated with Erlichadenocarcinoma cells (M ± m) Accumulation of ascitic The number of(weight Mean Group of animals injection gain, %) survival time Controlanimals 0 0.7 ± 0.1 9.6 ± 0.3 3 9.2 ± 1.2 7 28.4 ± 1.6 Intact animals 00.2 ± 0.1 35 ± 0 3 2.2 ± 0.3 7 5.1 ± 1.2 S-thioethylamine- 0 0.8 ± 0.116.2 ± 1.2 GSSG + normal 3 3.2 ± 0.6 saline 7 10.2 ± 2.2S-thioethylamine- 0 0.6 ± 0.1 15.2 ± 1.1 GSSG + 0.003% H₂O₂ 3 3.9 ± 0.67 9.2 ± 0.8 S-thioethylamine- 0 0.4 ± 0.2 17.2 ± 0.9 GSSG + 0.1% inosine3 3.2 ± 0.5 7 8.8 ± 1.1 S-thioethylamine- 0 0.5 ± 0.2 16.8 ± 1.2 GSSG +0.1% 3 3.7 ± 0.3 cystamine 7 9.6 ± 1.7 S-thioethylamine- 0 0.4 ± 0.117.2 ± 1.4 GSSG + 7% DMS0 3 4.8 ± 1.1 7 8.6 ± 1.1 Li salt of GSSG + 01.2 ± 0.3 12.2 ± 0.1 normal saline 3 8.1 ± 0.4 7 17.2 ± 1.4 Li salt ofGSSG + 0 1.5 ± 0.2 14.8 ± 0.6 0.003% H₂O₂ 3 6.9 ± 0.6 7 15.6 ± 1.6 Lisalt of GSSG + 0 1.4 ± 0.2 12.4 ± 1.4 0.1% inosine 3 9.1 ± 0.5 7 15.2 ±1.3 Li salt of GSSG + 0 1.7 ± 0.3 13.2 ± 1.2 0.1% cystamine 3 9.4 ± 0.27 16.6 ± 1.4 Li salt of GSSG + 7% 0 2.4 ± 0.2 13.2 ± 1.6 DMSO 3 9.8 ±1.0 7 17.6 ± 2.7 0.003% H₂O₂ 0 0.9 ± 0.2 9.8 ± 0.8 3 14.1 ± 0.6 7 27.2 ±1.2 0.1% inosine 0 0.8 ± 0.2 10.1 ± 0.6 3 11.9 ± 0.2 7 26.8 ± 2.1 0.1%cystamine 0 0.6 ± 0.2 10.2 ± 0.8 3 9.2 ± 0.8 7 25.8 ± 1.7 7% DMSO 0 0.5± 0.2 10.3 ± 1.8 3 11.4 ± 1.1 7 24.6 ± 2.2

TABLE 27 Effect of the lost artricles on the accumulation of asciticfluid and the mean survival time of mice inoculated with leukemia P388cells (M ± m) Accumulation of ascitic The number of (weight Mean Groupof animals injection gain, %) survival time Control animals 0 0.7 ± 0.121.2 ± 0.3 3 4.14 ± 0.9 7 18.2 ± 1.6 lntact animals 0 0.2± 0.1 35 ± 0 31.2 ± 0.3 7 4.8 ± 1.2 S-thioeethylamine- 0 0.7 ± 0.2 28.2 ± 0.8 GSSG +normal 3 2.1 ± 0.4 saline 7 8.2 ± 1.2 S-thioethylamine- 0 0.6 ± 0.2 29 8± 0.6 GSSG + 0.003% H₂O₂ 3 2.9 ± 0.4 7 6.6 ± 1.2 S-thioethylamine- 0 0.4± 0.3 31.2 ± 0.8 GSSG + 0.1% inosine 3 2.6 ± 0.6 7 6.8 ± 1.1S-thioethylamine- 0 0.5 ± 0.1 29.8 ± 2.8 GSSG + 0.1% 3 2.4 ± 0.4cystamine 7 6.6 ± 2.0 S-thioethylamine- 0 0.4 ± 0.1 22.2 ± 1.2 GSSG + 7%DMSO 3 2.6 ± 1.0 7 6.2 ± 1.4 Li salt of GSSG + 0 1.2 ± 0.2 32.2 ± 0.1normal saline 3 4.1 ± 0.4 7 11.2 ± 1.2 Li salt of GSSG + 0 1.5 ± 0.224.6 ± 0.6 0.003% H₂O₂ 3 4.9 ± 0.4 7 11.6 ± 1.7 Li salt of GSSG + 0 1.4± 0.3 28.2 ± 1.2 0.1% inosine 3 4.2 ± 0.6 7 8.8 ± 1.0 Li salt of GSSG +0 1.5 ± 0.1 26.8 ± 1.6 0.1% cystamine 3 3.4 ± 0.4 7 10.6 ± 1.7 Li saltof GSSG + 7% 0 2.4 ± 0.3 27.2 ± 1.4 DMSO 3 5.8 ± 1.0 7 8.6 ± 1.2 0.003%H₂O₂ 0 0.7 ± 0.2 21.2 ± 0.8 3 4.1 ± 0.4 7 19.0 ± 2.1 0.1% inosine 0 0.5± 0.2 21.8 ± 0.6 3 3.9 ± 0.4 7 16.6 ± 2.6 0.1% cystamine 0 0.4 ± 0.320.2 ± 0.3 3 5.2 ± 0.6 7 18.8 ± 1.1 7% DMSO 0 0.5 ± 0.1 20.6 ± 1.8 3 5.4± 1.4 7 19.6 ± 1.6

The most prominent antitumor effect in respect to slowdown of asciticfluid accumulation and prolongation of the mean survival time for eithertumor models (P388, L1210 leukemia and Erlich adenocarcinoma) wereobtained with S-thioethylamine-GSSG in combination with 0.1% inosine and7% DMSO.

EXAMPLE 15 Effect of Zinc Salt of GSSG and S-thioethylamine-GSSG andtheir Drug Forms on the Course of Experimental AllergicEncephalomyelitis (EAE), the Experimental Model of Multiple Sclerosis

The action of the zinc salt of GSSG in the combination withS-thioethylamine-GSSG in 0.003% solution of H₂O₂ (10 mg GSSG and 100 mgS-adenosyl-methyonine in 1 ml) and S-thioethylamine-GSSG in 5% solutionof ascorbinic acid were assessed in the model of EAE.

Within the frames of this study the influence of 10 day course of thenamed combinations on the cellular contents of blood (leukocyte,lymphocyte, monocyte, neutrophil count) was assessed. To assess cellularhypersensitivity to myelin basic protein and antigen of neuronalmembranes, in vitro migration activity of leukocytes in the peripheralblood was investigated in the presence of these antigens. We also usedcapillary method of reaction of inhibition of leukocyte migration(RILM). Neurological evaluation was carried out as well.

The study was done on male quinea-pigs with body weight 400-500 g.

Encephalitis—inducing substance—myelin basic protein (MBP) was obtainedfrom the bull spinal cord with the use of the method of columnchromatography and emulsified on complete Freund adjuvant. Immunizationof the animals was performed via inoculation of encephalitis-inducingmixture subcutaneously to the front paws (MBP+complete Freund adjuvant).Latency period of clinically expressed EAE averaged about 14-15 days,with minimun of 12 days.

Control Groups:

#1—Intact animals in which physiological solution was used;

#2—animals exposed to with encephalitis-inducing mixture who thenreceived physiological solution.

Experimental Groups:

#3—animals exposed to encephalitis-inducing mixture who then receivedzinc salt of glutation (GSSG-Zn) in the combination withS-adenosyl-methyonine in 0.003% solution of H₂O₂, 5 mg of GSSG base perkg of body weight.

#4—animals exposed to encephalitis-inducing mixture who then receivedS-thioethylamine-GSSG in 5% solution of ascorbinic acid, 5 mg of GSSGbase per kg of body weight

Neurological Evaluation (Scoring):

1 muscle weakness, discoordination of movements;

2 paresis of paws, urinary bladder atonia, urination disorders;

3 motor and functional palsies (pelvic organs);

4 blood circulation and thermoregulation disorders;

5 agony, Cheyne-Stocks breathing.

Methods for Cellular Immunity Evaluation

1. Reaction of Inhibition of Leukocyte Migration (RILM)—is a variant ofDelayed Hypersensitivity reaction in vitro. Basics of the method:changes in migration activity of peripheral blood leukocytes duringcontact with MB in glass capillaries. Migration zones are measured withocular micrometer of the microscope. Migration index is calculated asthe extent of migration of the cells with antigen to spontaneousmigration (without antigen) ratio. Statistically significant is thechange of the index by more than 0.2, ie migration index less than 0.8is considered suppression.

2. Spontaneous adhesion of blood leukocyte and its changes duringcontact with MBP. Migration of blood cells via vessel endothelium is akey moment in the development of inflammatory lesions in EAE. Thisprocess is determined by the combined action of adhesive molecules,expressed on leukocytes and endothelium. Suppression of adhesiveproperties of leukocytes by antigen indicates specific sensitization ofimmunized animals; changes in this parameter during inhibitioncharacterizes immunotropic properties of the drug towards cellularimmunity.

3. Adhesive activity of the cells is studied by the test of adhesion tomicropanels Falkon Plastic 3034 with fluorescent assessment of theresults and is expressed as the number of cells that adhere to thepanels spontaneously or after antigen exposure.

Calculation of the adhesion index:

(1—adhesion with antigen/spontaneous)×100 adhesion

Index>30 shows the reaction of suppression of spontaneous adhesion.

24 hours after the completion of the treatment with test articlessurvived animals were killed and the number of spleen cells was countedin sterile conditions. In the same time blood samples were taken toassess cellular counts.

Data reflecting the effects of the test articles on animal survival andneurological status are represented in tables 29 and 30. According tothese data, the use of GSSG-Zn and methyonil preparations helps increasesurvival of the animals and significantly decrease neurological symptomsof EAE (see tables 28 and 29).

In tables 30 and 31 the results indicating the influence of the testarticles on immunological EAE parameters are shown. The data demonstratea significant effect of the test articles on the parameters ofsensitization of blood lymphocytes to brain antigens. In the same timethere is a significant decrease in sensitization of lymphocytes in RILA(Table 30) and RILM (Table 31).

TABLE 28 Animal mortality during the experiment Total Died before theDied during the Total num- Animal number first injection of treatmentwith ber of ani- groups of animals the test articles the test articlesmals died ¹1 20 — — — ¹2 20 4 7 11  ¹3 20 5 — 5 ¹4 20 3 1 4

TABLE 29 Extent of neurological symptoms (score) Before the During theAnimal treatment with treatment with groups Number the test articlesNumber the test articles ¹1 20 — 20 — ¹2 16 3.06  9 3.22 ¹3 15 3.00 152.06 ¹4 17 3.10 16 2.50

TABLE 30 Parameters of sensitization of blood lymphocytes of quinea-pigswith EAE to brain antigens in RILA (%, before/after the treatment withthe test articles) Animal groups Antigen ¹1 ¹2 ¹3 ¹4 Myelin Basic —64.20 68.40 70.22 Protein 78.08 34.86 42.12 Neuronal — 50.10 48.14 54.76membrane 62.48 26.10 38.26 antigen

TABLE 31 Parameters of sensitization of blood lymphocytes of quinea-pigswith EAE to brain antigens in RILM (migration index, before/after thetreatment with the test articles) Animal groups Antigen ¹1 ¹2 ¹3 ¹4Myelin Basic 1.11 (±0.08) 0.52 (±0.10) 0.46 (±0.08) 0.54 (±0.12) Protein0.38 (±0.09) 0.88 (±0.11) 0.74 (±0.06) Neuronal 1.20 (±0.14) 0.68(+0.06) 0.60 (±0.04) 0.58 (±0.06) membrane 0.42 (±0.12) 0.92 (±0.06)0.82 (±0.10) antigen

In new Examples 16-26, the following truncated designations forchemically modified GSSG derivatives are used:

S-thioethylamine-glutathione disulfide S-thioethylamine- GSSGbis-[6,8-thioctanil] · glutathione disulfide bis-lipoil-GSSG[b-alanyl-hystidil] · glutathione disulfide carnosil-GSSG[-9-βD-ribofuranosyladenil] · glutathione disulfide adenosil-GSSGbis-[2-amino-4-[methylthio]butanoil] · glutathione bis-methionil-GSSGdisulfide

EXAMPLE 16 Therapeutical Effect of GSSG Series Preparations in Patientwith Severe Form of Acute Virus Hepatitis

A 32-year old male was hospitalized into infectious clinic with symptomsof pigment metabolism disorder; icterus of skin and mucous membranes;dark urine; light-colored faeces, high level of urobilinogen. Thephysical status of the patient was quite severe: body temperature −38.8°C., influenza-like and arthralgic syndromes. The patient's liver wasenlarged more than on 4-5 cm. Acute virus hepatitis was diagnosed. Thespecified diagnosis was acute virus hepatitis “B”. Severe form of thedisease with acute hepatic insufficiency.

The first course of the treatment with the use of GSSG seriespreparations comprised intravenous injections of GSSG-Na₂ in 0.1%solution of folic acid, once a day during 7 days at a dose of 0.1-0.5mg/kg of body weight.

The second course of the treatment comprised intravenous injections ofGSSG-Zn in 0.1% solution of inosine once a day during 7 days at a doseof 0.1 mg/kg of body weight.

The third course comprised intravenous injections of GSSG-Na₂ in 5%solution of ascorbic acid every other day during 7 days at a dose of0.1-0.5 mg/kg of body weight. Simultaneously the same formulation wasinjected intramuscularly at the same dose one time a day.

The data in table 32 show the beneficial changes hematology/immunologyparameters, decrease in (or/and normalization of) hepatitis laboratorymarkers. Thus, elimination of pathologic process and the patient'srecovery occurred to be 1.5-2 months earlier than ordinary period ofrecovery in acute virus hepatitis.

TABLE 32 Changes in hematological, immunological, serological andbiochemical parameters during 24 days after beginning the treatment withthe use of GSSG series preparations 24 days after Prior to the thetreatment Parameter treatment beginning Hematology/ImmunologyErythrocytes 3.65 × 10¹²/L × 10¹²/L Hemoglobin 96 g/L 128 g/L Leukocytes12.6 × 10⁹/L 7.8 × 10⁹/L Lymphocytes 1.1 × 10⁹/L 2.9 × 10⁹/L Platelets180 × 10⁹/L 240 × 10⁹/L ESR 28 mm/hour 12 mm/hour CD3⁺ 711 × 10⁸/L 1319× 10⁶/L CD4⁺ 410 × 10⁸/L 655 × 10⁶/L CD8⁺ 305 × 10⁸/L 662 × 10⁶/LCirculate Immune Complexes 142 units 108 units IFNα 368.0 pg/mL 906.0pg/mL IFNγ 102.0 pg/mL 608.0 pg/mL IL-6 245.9 pg/mL 653.0 pg/mL SerologyHbs Ag + − Hbe Ag + − IgM Anti Hbs + + Anti Hbe − + Anti Hbs Ag − ±Blood Chemistry Bilirubin -total 180 μmol/L 28.5 μmol/L -direct reacting31 μmol/L 6.1 μmol/L ALT 910 U/L 90.8 U/L AST 208 U/L 22 U/L Alkalinephosphatase 1316 U/L 206 U/L Serum cholinesterase 24 μmol/L 268 μmol/Lγ-Glutamyl transpeptidase 205 IE/L 39 IE/L Prothrombin Index of p 38%87% Acid-base balance metabolic normal alkalosis

EXAMPLE 17 Therapeutical Effect of GSSG Series Preparations in Patientwith Chronic Virus Hepatitis in Stage of Exacerbation

A 56-year old female was hospitalized into infectious clinic withcomplaint of physical status worsening, weakness, fatigue andirritability, anorexia, nausea. She also had some GIT symptoms, pain inright sub-costal area and body temperature up to 38.5° C.

On examination: The general status of the patient was of middleseverity. Palpation revealed splenomegaly and hepatomegaly. Liverprotruded 3 cm from under the rib arch. Sclerae and mucous membraneswere subicteric.

Ultrasonic examination: Liver was remarkably enlarged, v. portae was 15mm, the gall-bladder wall was thickened, pancreas had normal structure,spleen was enlarged (578×168 mm) and thickened. Kidneys were withoutremarked alterations of structure.

During 2 weeks the patient was receiving desintoxication and antiviraltherapy (Roferon-A, a recombinant α₂-interferon). Because of a furtherdisease progress and inefficacy of the therapy used, the decision wastaken to try the treatment with GSSG series preparations.

Intravenous injections of GSSG-Na₂ in 10% solution of choline-chloridewere applied during 7 days, (once a day at a dose of 0.1-1.0 mg/kg ofbody weight).

Intravenous injections of bis-methionil-GSSG in 5% solution of ascorbicacid were applied during the subsequent 10 days (once a day at a dose of0.1-0.5 mg/kg of body weight). Simultaneously, intramuscular injectionsof GSSG-Na₂ in 0.1% solution of inosine were applied during these 10days (once a day at a dose of 0.01-0.5 mg/kg of body weight).

One month after the aforementioned treatment with GSSG seriespreparations the patient's physical condition improved significantly.Only the complaints of weakness and fatigue remained. Diminution inalgetic and dyspeptic syndromes was remarkable, body temperature wasnormal. Some beneficial changes in laboratory indices were observed (seetable 2). Ultrasonic examination revealed the same extent ofsplenomegaly and hepatomegaly.

Due to the certain improvement in the patient's status, but remaining ofhepatic lesion signs the decision was taken to apply another course oftreatment with the use of GSSG series preparations. After the secondcourse of the completely the same treatment the patient had nocomplaints. Pain and dyspeptic signs were absent. Ultrasonic examinationrevealed diminution in spleen and liver dimensions. Palpation revealedliver protruded 1.5 cm from under the rib arch. (For laboratory data seetable 33).

Thus, the application of combined therapy with the use of GSSG seriespreparations conditioned a remarkable curative effect, with improvementin physical status and life quality, stabilization and reversion of thepathologic process, beneficial laboratory changes.

TABLE 33 Changes in hematological, immunological, serological andbiochemical parameters after the 1^(st) and 2^(nd) courses of thetreatment with the use of GSSG series preparations Prior to After the1^(st) After the 2^(nd) the course of the course of the Parametertreatment treatment treatment Hematology Erythrocytes 10¹²/L 3.79 4.34.85 Hemoglobin g/L 128 126 133 Leukocytes 10⁹/L 3.6 3.9 5.6 Lymphocytes10⁹/L 900 1600 2900 Platelets 10⁹/L 140 156 220 ESR mm/hour 23 18 11Immunology A-lymphocytes (ND20⁺)10⁶/L 270 302 389 ND4⁺ 10⁶/L 374 407 936ND8⁺ 10⁶/L 204 612 727 CD25⁺ 10⁶/L 228 246 680 Circulating ImmuneComplexes 190 170 106 IgA, g/L 5.6 5.2 1.2 IgM, g/L 6.9 6.0 4.8 IgG, g/L29.0 18.9 3.4 IFNα pg/mL 304.6 200.8 128.6 IFNγ pg/mL 215.2 110.0 78.1IL-1β, pg/mL 405.5 198.0 158.9 IL-6, pg/mL 603.9 430.6 190.2 SerologyHBs Ag + + − anti-HBs − + + anti-HBs-IgM − + + HBeAg + − − anti-IAe− + + Blood Chemistry Bilirubin -total μmol/L 46.0 28.4 20.8 -directreacting μmol/L 27.0 15.7 6.2 ALT U/L 9.1 4.3 1.2 AST U/L 0.8 0.7 0.5Alkaline phosphatase U/L 12.6 8.2 7.8 γ-Glutamyl transpeptidase IE/L 208190 41 Total protein g/L 91 89 78 Albumin, % 40 44 60 α₁-globulin, % 6.26.4 5.2 α₂-globulin, % 7.4 7.5 9.0 β-globulin, % 16.82 16.2 12.6γ-globulin, % 29.58 25.9 13.2

EXAMPLE 18 Therapeutical Effect of GSSG Series Preparations in Patientswith Acute Peritonitis

1. A 78-year old female was hospitalized with diagnosis of incarceratedventral hernia, acute intestinal obstruction with necrosis of the smallintestine and developing toxic phase of acute peritonitis. During thesurgical operation approximately 800 ml of feculent effusion with flakesand fibrin clots was found in and removed from the abdominal cavity.Incarcerated part of small intestine was necrotized.

A partial resection of the small intestine with creating anastomose wasperformed. A probe was conducted into the small intestine through theesophagus and stomach and placed to ileocecal angle. Duringpostoperative period the evacuation of intestine contents was performedonce a day. After that 150 ml of 0.1% solution of folic acid in dimethylsulfoxide (DMSO), comprising 10 ml of 1% GSSG disodium salt solution wasinstillated into the small intestine. Simultaneously 0.1% solution offolic acid and 1% GSSG disodium salt solution was injected intravenouslyat a dose from 0.01 to 0.1 mg/kg once a day during the 4 days.

An adequate peristalsis restored on the 2^(nd) day after surgery. Theprobe was removed on the 8^(th) day. The patient was checked out fromthe hospital on the 19^(th) day.

2. A 16-year old male was hospitalized with a diagnosis of acutecommissure-induced obstruction of small intestine with development ofthe peritonitis; toxic phase.

During the operation approximately 600 ml of feculent effusion withflakes and fibrin clots was found in and removed from the abdominalcavity. The patient had a prominent commissure process. A commissurotomywas made. A probe was conducted into the small intestine through theesophagus and stomach and placed to ileocecal angle.

During postoperative period the evacuation of bowel contents wasperformed once a day. After that 150 ml of 0.1% solution of folic acidin dimethyl sulfoxide (DMSO), comprising 10 ml of 1% GSSG disodium saltsolution was injected into the small intestine. Simultaneously 0.1%solution of folic acid and 1% GSSG disodium salt solution was injectedintravenously at a dose from 0.01 to 0.1 mg/kg once a day during the 4days.

An adequate peristalsis renewed on the 2^(nd) day, and probe was removedon the 8^(th) day after surgery. The patient was checked out from thehospital on the 11^(th) day.

EXAMPLE 19 Therapeutical Effect of GSSG Series Preparations in Patientwith Cancer of the Prostate

A 63-year old male was hospitalized in urology department with thesuspicion of prostate cancer. Palpation revealed the enlarged, denseprostate. X-ray examination of thorax revealed metastasis in frontalparts of IV-VII ribs, as well as in cranium, vertebrae, pelvic andfemoral bones. Cancer of prostate was diagnosed with multiple bonemetastasis (T₂N₂M₁).

Course of treatment was applied during the 30 days and was conducted bythe following scheme:

1. Intravenous injections of GSSG-Zn in 1% solution of inosine were madeduring 10 days once a day at a dose of 0.01-0.5 mg/kg of body weight. Inall cases where Zn salts are used in the Examples of this application,two atoms of Zn are present with one atom between X₁ or X₄ and one atombetween X₂ or X₃.

2. During the next 10 days injections of S-thioethylamine-GSSG were madeendolymfaticaly once a day at a dose of 0.1-1.0 mg/kg of body weight.

3. During the next 10 days intravenous injections of GSSG-Zn in 1%solution of inosine were made every other day at a dose of 0.01-0.5mg/kg of body weight.

After the treatment course the patient's condition improvedsignificantly, pain in groin area became of less intensity, episodes offrequent urination reduced up to 2-3 times a night, edema of lowerextremities decreased. Hematological examination revealed somebeneficial changes (table 34).

After checking out the patient received intramuscular injections ofGSSG-Zn in 1% solution of inosine twice a week at a dose of 0.01-0.5mg/kg of body weight.

More than 1 year later the patient was hospitalized again at the sameurology department for examination and conducting of the repeated coursewith GSSG series preparations. The treatment scheme with the use of theGSSG series preparations was identical. The main therapeutical effects 3months after the end of the second treatment session was considered asthe following: Improvement in life quality; absence of lower extremityedema; regression of enlarged lymph nodes; absence of disurymanifestations; decrease in size of prostate; beneficial X-ray dynamics(calcification of some particular metastasis in ribs and vertebrae);restoration of hematology and immunology indices.

TABLE 34 Variations of hematology and blood chemistry indices 1 month 2months Prior after the after the to the treatment treatment Parametertreatment beginning beginning Erythrocytes, 10¹²/L 3.8 3.9 4.0Hemoglobin, g/L 110 114 128 Leukocytes, 10⁹/L 7.9 5.4 5.0 Stubneutrophils, % 4 4 2 Segmented neutrophils, % 75 57 38 Lymphocytes, % 1627 41 Monocytes, % 3 12 6 ESR, mm/hour 38 28 12 Platelets, 10⁹/L 168 225244 ÑD3⁺, % 32.6 44.8 72.2 ÑD4⁺, % 16.1 22.8 50.2 ÑD8⁺, % 11.0 15.4 16.9“Active” Ó-lymphocytes (bearing 12.9 60.2 62.4 receptors to IL-2, CD25⁺)NÊ-cells (CD16⁺/ÑD56⁺), μL⁻¹ 64 292 404 Â-lymphocytes (CD20⁺), % 6.410.2 15.4 Circulating Immune Complexes, units 385 212 102 Creatinine,mmol/L 0.06 0.08 0.07 Acid phosphatase, U/L 324 218 164 Alkalinephosphatase, U/L 10.4 8.3 6.8 Total bilirubin, mmol/L 17.8 8.5 9.0Glucose, mmol/L 4.8 4.3 4.9

EXAMPLE 20 Therapeutical Effect of GSSG Series Preparations in Patientwith Cancer of the Pancreas

In May 1996 56 year old male patient was admitted to the II surgicaldivision of the Hospital N122. On admission the patient's condition wassevere. The patient complained on: continuous band-like pain in theupper part of the abdomen, that worsened when the patient was lying onthe back, appetite loss, nausea, vomiting, flatulence, diarrhea.Karnofsky index 40. On palpation: pain and tension of the abdominalmuscles at the site of projection of the pancreas (Kerte's symptom),Pancreas was solid, with uneven surface, enlarged. The liver was solid,4 cm below the lower rib.

On ultrasonic examination: pancreas was enlarged (head 7 cm, body 4 cm,tail 2.5 cm) with uneven borders, solid. Virsung duct was enlarged 0.7cm. Liver was enlarged, right lobe 18 cm, left lobe 10 cm. Lower edgewas rounded. Its edges were uneven, the structure was nonhomogeneouswith multiple hyperechogeneous foci in the parenchyma (metastases).Diagnosis—cancer of the pancreas with liver metastases (T3N2M1).

Treatment: detoxification, protease inhibitors, narcotic analgesics.

Due to the fatal severity of the patient's condition and absence of anyalternative treatment the decision was made to use treatment with GSSGseries preparations.

The treatment scheme: zinc salt of GSSG (0.01-0.5 mg/kg) intravenously(every day twice a day for 10 days). For the next 10 days—zinc salt ofGSSG (0.01-0.5 mg/kg/day) intravenously, every other day and zinc saltGSSG in 7% dimethyl sulfoxide (0.1-1.0 mg/kg/day) endolymphatically. Forthe next 10 days (third decade) zinc salt of GSSG (0.01-0.5 mg/kg/day)intravenously, twice a week.

After a month treatment patient's condition was moderately severe;periodical moderate pain in the left subcostal region. Kamofskyindex—60. Narcotic analgesics were stopped, appetite increased. Therewas a tendency to improvement in the blood parameters.

During 3 weeks after the completing intravenous treatment course thepatient was receiving zinc salt of GSSG (0.01-0.5 mg/kg) once a week,intramuscularly.

In June 1996: the patient was again admitted to the 11 surgical divisionof the hospital 122 for investigation and for conducting a new sessionof treatment with the use of GSSG series preparations.

On examination: marked improvement of the clinical condition and bloodparameters (see Table 4). The patient's condition was almostsatisfactory. Complains on a periodic weak pain in the left subcostalregion. Karnofsky index—70. On palpation—some tenderness in the site ofthe projection of the head and body of the pancreas; the organ was lesssolid with a smoother surface.

On ultrasonic examination: some decrease in the size of pancreas (head6.3 cm, body 3.2 cm, tail 2.1 cm). Virsung duct 0.4 cm. The liver wasenlarged; the right lobe 16.5 cm, the left one—9.5 cm. Contours of theliver were uneven, structure—nonhomogeneous. Fibrosis of the portahepatis. Multiple hyperechogeneous shadows in the liver parenchyma.

The treatment scheme for the second session of therapy with GSSG seriespreparations was the following. For the first 10 days,S-thioethylamine-GSSG was infused every day via catheter into thehepatic artery (0.1-1.0 mg/kg/day).

After that the zinc salt of GSSG (0.01-0.5 mg/kg) was givenintravenously every day, for 10 days. Being checked out the patientreceived supportive therapy with the zinc salt of GSSG intravenously(0.01-0.5 mg/kg/day), once a week, for 1 month.

In September 1996 the patient was again admitted to the Hospital N122for examination and the third session of therapy. Clinical condition didnot change by that time. For blood parameter variations see table 35.

On ultrasonic examination: pancreas had the same characteristics asbefore. Liver was slightly enlarged, the right lobe—15 cm, the leftlobe—8 cm, lower edge rounded, edges were definite and smooth,parenchyma was nonhomogeneous due to hypo and hyperechogeneous foci(fibrosis and calcificates).

Thus the following clinical effects were considered as prominent andsignificant: improvement of the quality of life; stabilization of theneoplastic process; resolution of some metastases; restoration ofimmunology and hematology indices; improvement in results of ultrasonicexamination.

TABLE 35 Laboratory indices in patient with pancreas cancer throughoutthe observation period After one After 3 After 6 Before month of monthsof months of Parameter treatment treatment treatment treatment Normallimits Hematology and blood chemistry Erythrocytes, 10¹²l 3.8 4.0 4.14.6 4.0-5.0 Hemoglobin, g/l 128 130 134 141 130.0-160.0 Platelets, 10⁹/l216 226 234 268 180.0-320.0 Leukocytes, 10⁹/l 9.6 8.8 8.3 6.8 4.0-9.0Neutrophils and rods, % 10 4 3 3 1-6 Segmentonuclear neutrophils, % 7059 50 54 47-72 Lymphocytes, % 15 27 39 34 19-37 Monocytes, % 4 6 6 7 3-11 Eosinophils, % 6 4 2 2  0-11 ESR, mm/h 64 30 19 15  2-10 Totalprotein g/l 74 72 69 71 65-85 ALT/mmol/hL 0.8 0.4 0.33 0.3 0.1-0.7AST/mmol/hL 0.5 0.4 0.21 0.2 0.1-0.5 α-amylase g/hL. 46 30 21 18 12-32LDH MU/l 1121 542 521 472 <450 Immunology Lymphocytes 1440 2376 32372312 1200-3000 B-lymphocytes (CD20+) 10⁶/l 272 348 554 392 200-400T-helpers (CD4+), 10⁶/l 874 1114 1242 1092  700-1100 T-suppressors(CD8+) 10⁶/l 222 384 1082 721 500-900 CD4+/CD8+ ratio 3.94 1.63 1.151.51 1.0-1.5 Circulating immune complexes 362 194 136 108  50-100

EXAMPLE 21 Therapeutical Effect of GSSG Series Preparations in Patientwith Diabetes Mellitus

Female patient of 18 year old was admitted to the Division ofEndocrinology of the Hospital N16. Diagnosis: insulin dependent diabetesmellitus (type I). Severe form of insulin resistance, diabeticangioretinopathy grade IV, initial symptoms of diabetic polyneuropathyIII from the age 14. The disease started with precomatose state, bloodglucose varied from 18.4 to 28.0 mmol/l, ketoneuria (acetone), andglucosuria up to 12%. Total Insulin dose at the disease onset:SU-Insulin 68 units, “ICS” 269 units. Previously was hospitalizedseveral times. Dose of insulin reached 500 units in 1994-1996. At thattime blood glucose was 19.6-24.3 mmol/l, glucosuria was 4-6%, positiveketones in urine.

In September 1996 the patient was admitted to consider modification oftreatment with the dose of ICS insulin 500 units, ICS-A 100 units,SU-Insulin 5-units. On admission Insulin dose was the following:B-insulin 480 units, SU insulin 22 units.

Blood sugars:

9 A.M. 11.4 mmol/l noon 11.1 mmol/l 2 P.M. 13.9 mmol/l 5 P.M. 16.7mmol/l 6 A.M. 10.7 mmol/l

Glucosuria—up to 670 mmol, ketones +++.

Due to the severity of the patient's insulin resistance and after thepatient's agreement the decision was made to use GSSG seriespreparations.

Treatment scheme: intravenously for 10 days, once a day GSSG-Na₂ in 0.5%solution of lipoic acid (0.01-0.5 mg/kg/day).

For the next 10 days, intravenously, every other day—zinc salt GSSG in0.5% solution of lipoic acid (0.01-0.5 mg/kg/day).

For the next 10 days (third decade) sodium salt GSSG-Na₂ in 5% solutionof ascorbic acid, intramuscularly, once a day for 20 days (0.1-0.5mg/kg/day).

After the treatment session had been completed the insulin treatment wasmodified. SU insulin was started, several injections per hour, sustainedrelease insulin canceled.

The dose of insulin was gradually decreased, and the patient wasdischarged with the following insulin regimen:

6 A.M. 4 units SU-insulin 9 P.M. 50 units 2 P.M. 36 units 7 P.M. 36units 11 P.M. 8 units (total dose 134 units).

Blood sugars got to normal and did not exceed 8 mmol/l even after meals.After the patient was discharged she received therapy with GSSG seriespreparations as an out-patient for a month. Treatment scheme was thefollowing:

During a month—sodium salt of GSSG in 0.5% solution of lipoic acid(0.01-0.5 mg/kg), intramuscularly, every other day.

During the 2 successive months the patient had two episodes ofhypoglycemia (1.8-2.2 mmol/l), and that forced to decrease insulindoses. Thus, after the two months of treatment following insulin regimenwas implemented:

6 A.M. 4 units SU-insulin 9 P.M. 36 units 2 P.M. 12 units 7 P.M. 12units 11 P.M. 4 units (total dose 68 units).

The patient's condition was satisfactory, no complains. See table 36;for changes in blood parameters. Thus the following clinical effectsseem to be substantial: stopped insulin resistance; decreased insulindose, improvement of the quality of life and laboratory parameters.

TABLE 36 Hematology, blood chemistry and immunology parameters inpatient with juvenile diabetes mellitus After one After 2 Before monthof months of Parameter treatment treatment treatment Erythrocytes,10¹²/l 4.1 4.3 4.4 Hemoglobin, g/l 129 135 144 Platelets, 10⁹/l 205 222278 Leukocytes, 10⁹/l 7.8 6.4 5.2 Neutrophils and rods, % 4 3 3Segmentonuclear neutrophils, % 39 53 58 Lymphocytes, % 51 39 34Monocytes, % 4 3 4 Eosinophils, % 2 2 1 ESR, mm/h 13 12 10 ALT/mmol/hL0.44 0.38 0.22 AST/mmol/hL 0.3 0.3 0.3 Total protein, g/l 75 72 72Bilirubin, total, mcmol/l 10.8 9.2 8.4 Cholesterol, total, mcmol/l 7.46.54 5.8 Triglycerides, mcmol/l 4.2 3.5 2.1 Urea, mmol/l 4.2 4.0 3.3Creatinine, mmol/l 0.133 0.095 0.088 B-lymphocytes (CD20+) 10⁶/l 478 395388 T-helpers (CD4+), 10⁶/l 1412 1014 874 T-suppressors (CD8+) 10⁶/l1044 942 605 CD25⁺ 422 512 495 Circulating immune complexes 214 123 95

EXAMPLE 22 Therapeutical Effect of GSSG Series Preparations in Patientwith Lung Cancer

Diagnosis: Cancer of the upper lobe of the right lung (T3N2M0) extendinginto superior caval vein and pulmonary artery. Metastases to mediastinallymph nodes.

In September 1995 a male patient of 59 was admitted to the surgicaldivision of the Institute of Pulmonology with suggested lung neoplasm.On admission the patient was complaining of periodical fever up to 38.4°C., decreased appetite, 8 kg weight loss, dyspnea after movement.

On several chest CT scans (29.08.96) the size of the right lung wasdecreased due to hypoventilation of the upper lobe. Pulmonary treelooked different on the right side. Right upper lobe bronchus wasnarrowed and deformed, with a focal shadow 4.0×5.0×6.0 cm. Thereprobably was an enlargement of paramediastinal lymph nodes. No fluid inpleural cavities.

On bronchoscopy: lymphangiitis of the lateral wall of the lower 1/3 oftrachea, lymphangiitis and infiltration of the lateral wall of the rightmain bronchus, compressive and infiltrative stenosis of the right upperlobe bronchus (lateral wall) of the 1-2 degree.

On diagnostic thoracotomy: (14.09.95) uneven dense formation of theupper lobe of the right lung 4.0×5.0×6.0 cm, growing into upper parietalpleura. On intraoperative examination, after pericardium was opened itbecame obvious that neoplasm extended into superior caval vein andpulmonary artery. Histologically, neoplasm was identified as lowdifferentiation (small cell) carcinoma. The case was consideredinoperable.

The patient's state continued to deteriorate: right supraclavicular nodeenlarged (3.5×4.0 cm). The patient was transferred to the Institute ofOncology for chemotherapy. After the first treatment course ofCisplatine combined with Etoposide the patients' condition deterioratedsignificantly: nausea and vomiting, alopecia, increased transaminases,creatinine, leukopenia.

Due to the severity of the patient's disease and absence of anyalternative treatment the decision was made to use compassionatetreatment with lithium salt of GSSG.

From the first injections of GSSG-Li (0.01-0.5 mg/kg per day,intravenously/intramusculariy alternative days for 14 days), the qualityof life improved significantly (Kamofsky index 80 (60), appetiteincreased, dyspnea decreased.

After 2 weeks' treatment with lithium salt of GSSG in 0.003% solution ofH₂O₂ blood indices substantially improved (leukocyte count, red cellcount, creatinine, transarbinases), and that permitted to try anotherchemotherapy course (Cisplatine combined with Etoposide). Compared withthe first course, there were no complications during the second one whenthe patient was receiving U-GSSG: there was no nausea, vomiting, therewas an increase in appetite (5 kgs weight gain); results of clinical andbiochemical blood tests were within normal limits.

After the second chemotherapy course therapy with GSSG seriespreparations was resumed: lithium salt of GSSG in 3% of dimethylsulfoxide (0.01-0.5 mg/kg per day intravenously/intramascualarly 3 timesa week during 14 days).

Then the patient received another chemotherapy course in the Instituteof Oncology. There were no complications during the third chemotherapycourse. Result of blood tests were within normal limits. There were acomplete resolution of the enlarged right supraclavicular lymph node andX-ray demonstrated improvement of the primary lesion; no signs ofatelectasis, additional shadows were neither found in mediastimun, norparatracheally; trachea had a normal location; there was no residualcavity.

Thus the 2 courses of chemotherapy combined with GSSG seriespreparations (lithium salt of GSSG) led to satisfactory quality of life,systemic regression of the neoplasm, restoration of laboratoryparameters which was associated with a stable increase in endogenousproduction of cytokines and hematopoietic factors (see Table 37). Lateron the patient was receiving GSSG-Li₂ at a dose rate 0.01-0.5 mg/kg,every other day for 14 days a month during a year.

TABLE 37 Laboratory parameters in patient with lung cancer throughoutthe observation period After two After 4 After Before months of monthsof one Parameter treatment treatment treatment year Normal limitsErythrocytes, 10¹²/l 3.9 4.4 4.7 4.5 4.0-5.0 Hemoglobin, g/l 120 132 135142 130.0-160.0 Platelets, 10⁹/l 396 235 282 270 180.0-320.0 Leukocytes,10⁹/l 3.1 4.1 5.5 6.3 4.0-9.0 Neutrophils and rods, % 2 3 2.5 4 1-6Segmentonuclear neutrophils, % 74 41 42 47 47-72 Lymphocytes, % 16 40 3635 19-37 Monocytes, % 6 9 12 10  3-11 Eosinophils, % 1 5 5.5 4  0-11ESR, mm/h 34 21 12 9  2-10 Total protein, g/l 61 78.4 80.6 78 65-85Albumin, % 35.96 49.0 61.6 56 50-66 α1-globulins, % 10.32 4.0 2.8 4.72.5-5.0 α2-globulins, % 15.24 12.0 7.2 9.2 6.0-9.5 β-globulins, % 15.2413.6 12.8 12.8  8.0-13.0 γ-globulins, % 23.33 21.4 15.6 17.3 13.0-17.0A/G ratio 0.56 0.84 1.6 1.27 1.0-1.9 Urea, mmol/l 9.0 6.2 6.3 5.92.5-8.3 Creatinine, mmol/l 1.21 0.88 0.89 0.88 0.044-0.115 Bilirubin,total, mcmol/l 19.5 13.0 14.0 5.1  3.5-20.5 Prothrombin index, % 88 90104 100  80-105 Glucose, mmol/l 7.1 5.4 5.3 4.6 3.3-6.1 ALT/mmol/hL 1.320.21 0.19 0.19 0.1-0.7 AST/mmol/hL 1.22 0.36 0.15 0.17 0.1-0.5Lymphocytes 496 1640 1980 2205 1200-1300 β-lymphocytes (CD20+) 10⁶/l 101232 392 372 200-400 T-helpers (CD4+), 10⁶/l 321 824 1020 1064  700-1100T-suppressors (CD8+) 10⁶/l 74 484 654 608 500-900 CD4+/CD8+ ratio 4.31.7 1.55 1.75 1.0-1.5 Cells with IL-2 receptors 202 472 682 605 208-576(CD25+)/10⁶/l HLA (11) receptor, 10⁶/l 294 392 472 541 304-720 NK-cells(CD16+) 10⁶/l 180 320 525 394 200-400 IgA, g/l 3.28 4.01 5.1 4.8 0.8-5.2IgM, g/l 0.5 0.71 1.4 2.1 0.6-3.8 IgG, g/l 13.8 15.4 17.4 16.5  6.0-18.0Circulating immune complexes 335 221 112 62  50-100 IL-1β, pcg/ml 19.8178.6 294.8 132 TNF α, pcg/ml 34.1 121 149 98

One Year Later (14.10.96)

On chest CT scan there was a site of fibrous changes in the right upperlobe bronchus 1.5×1.5×2.0 cm. There were no new Infiltrative or fibrouschanges in lung tissue. Lumens of the trachea and bronchi in both lungswere neither narrowed nor deformed. There was no mediastinal and lungroot lymph node enlargement. No fluid in pleural cavities.

CONCLUSION

Combined treatment with chemotherapeutic regimens and GSSG-Li₂ producedremarkable potentiation of the chemotherapeutic effect with itsacceptable chemotherapy tolerability. Generally, the effect of thecombination led to tumor regression, liquidation of metastases,restoration of immune and hematopoietic systems, a better quality oflife.

EXAMPLE 23 Therapeutical Effect of GSSG Series Preparations in Patientwith Sigmoid Cancer

Diagnosis: Cancer (undifferentiated adenocarcinoma) of the rectosigmoidregion of the colon (T4N0M0), complicated by right-sided adnextumor,right-sided tuboovarial abscess, diffuse phlegmonous-purulentperitonitis.

In June 1996 a female patient of 48 was urgently admitted to the II^(nd)surgical division of the Central Hospital N122 with the above mentioneddiagnosis.

Operation: loop sigmostoma, abdominal cavity cleaning and draining. Thepatient's condition was severe: Karnofsky 30, fever 39-40° C. Despiteaggressive therapy clinical condition continued to deteriorate andcomplicated with bilateral pneumonia. Additional intensive therapy withantibiotics (Cefalosporins and Penicillins—Claforan 6 g/day,Ampicillin/Oxacillin 2 g/day) did not result in desirable effect.

Due to inefficacy of the treatment the decision was made to usecompassionate treatment with GSSG-Zn₂.

For one week the patient received GSSG-Zn₂ in 0.1% solution of cystamine0.01-0.5 mg/kg per day IV and IM together with the previous complextherapy. After one weeks' treatment the patient's physical statusimproved substantially, appetite increased, weakness disappeared, sleepnormalized, local healing of the wound accelerated. There was a tendencyfor normalization of blood parameters (see Table 38), there were noX0ray signs of pneumonia.

Those positive effects permitted to perform the second stage of thesurgical treatment—to repeat laparotomy with resection of proctosigmoidregion of colon and right adnexa with ovary. Postoperative period wascharacterized by right-sided lower lobe pleuropneumonia. Again, GSSG-Zn₂in 0.1% cystamine solution was added to the treatment (0.01-0.5 mg/kgevery day intravenously and intramuscularly during a week). Clinical andradiological signs of pneumonia resolved within 3 days.

There were no complications of the postoperative wound. Sutures wereremoved on the seventh day. Patient was discharged in satisfactorycondition to be followed up as an out-patient.

General effects can be summarized as follows: improvement of the qualityof life; potentiation of antibacterial therapy; restoration oflaboratory indices; acceleration of wound-healing; possibility ofradical surgical treatment.

TABLE 38 Laboratory parameters in patient with sigmoid cancer throughoutthe observation period After one After two Before week of weeks of Afterone Parameter treatment treatment treatment month Erythrocytes, 10¹²/l3.2 3.8 3.8 4.1 Hemoglobin, g/l 112 126 130 132 Platelets, 10⁹/l 200 220274 248 Leukocytes, 10⁹/l 24.0 9.1 8.2 7.8 Neutrophils and rods, % 13 66 5 Segmentonuclear 66 60 58 50 neutrophils, % Lymphocytes, % 12 26 2836 Monocytes, % 3 5 5 7 Eosinophils, % 6 2 2 2 ESR, mm/h 62 18 15 12ALT/mmol/hL 0.8 0.5 0.3 0.22 AST/mmol/hL 0.5 0.4 0.4 0.18 IgA, g/l 3.83.6 3.5 3.2 IgM, g/l 2.8 2.5 3.0 2.8 IgG, g/l 19.8 16.3 17.4 Circulatingimmune 162 134 94 96 complexes

EXAMPLE 24 Therapeutical Effect of GSSG Series Preparations in Patientwith Pancreas/duodenum Cancer

Diagnosis: Cancer of the pancreas extending into the duodenum (T3N2M1).

In January 1996 a male patient of 67 was admitted to the II surgicaldivision of the Hospital N122 with obstructive jaundice.Diagnosis—infiltrative stricture of the main bile duct. Surgery:transcutaneous-transhepatic external-internal drainage.

In February 1996—cholecystectomy, choledocho-duodenoanostomosis (Jurashprocedure). Patient was suffering from severe pain in the rightsubcostal area irradiating to the back, and narcotic analgesics wereprescribed. No appetite. Weight loss 13 kg per month. Karnofsky index40. Periodical nausea, vomiting, steatorrhea. Increased amylase andlipase in blood serum, amylase in urine, anemia. In the projection ofthe head of the pancreas turnorous object could be palpated. Onfibrogastroduodenoscopy: infiltration of duodenal mucosa down to theFater's papilla.

Due to the severity of the patients disease, progressive deteriorationand absence of any alternative treatment the decision was made to usecompassionate treatment with GSSG-Zn₂ in dimethyl sulfoxide (DMSO).

After 2 weeks' treatment the patient's condition obviously improved.Kamofsky index—80. Continuous pain decreased, though the patient wassuffering from periodical moderate pains, narcotics were canceled. Nonausea or vomiting; weight gain 4 kg; improvement in blood parameters(see Table 39).

Clinical and laboratory improvement permitted to introduce combinedtreatment with high-dose chemotherapy (5-Fluoruracil-10 g/course) whichwas combined with GSSG-Zn₂. During 3 days the patient was receivingFluoruracil endolymphatically (day 1-3 g, day 2-3 g, day 3-4 g) and highdoses of GSSG-Zn₂ in DMSO (0.1-1.0 mg/kg per day).

The patient tolerated treatment without hematological and other toxiccomplications. (for blood parameters see Table 40). During the 3consecutive months (February-April) the patient received supportingdoses of GSSG-Zn₂ in DMSO (0.01-0.3 mg/day, intravenously andintramuscularly, 2 times a week). Patient's condition was quite good;Kamofsky index 90. No pains; good appetite; weight gain—8 kg.

3 months later (May, 1996), a 3 day course of high-dose chemotherapytogether with GSSG-Zn₂ was conducted (total dose of fluoruracil—10 g;GSSG-Zn₂ 0.1-1.0 mg/kg, daily) without complications reported. Thepatient was discharged in satisfactory condition to be followed up as anout-patient. Treatment with GSSG-Zn₂ was continued for 3 successivemonths (0.01-0.5 mg/kg IV and IM 2 times a week).

6 months later (September 1996): the third course of high-dosechemotherapy according to the scheme shown above, with no complications.Patient's condition was satisfactory; Kamofsky index 90. Good appetite;no nausea or vomiting; Weight gain 12 kg from the beginning of theobservation. On gastroduodenoscopy—significant decrease in infiltrationof duodenal mucosa with signs of stabilizing the neoplastic process.

Thus the following effect of the combination of GSSG-Zn₂ andchemotherapy regimen can be stressed: improvement of the quality oflife; resolution of pain; beneficial laboratory changes; stabilizationof the neoplasm process; good tolerance of chemotherapy.

TABLE 39 Laboratory parameters in patient with pancreas/duodenum cancerthroughout the observation period After one After six Before After twoweeks months of After 3 months months of Parameter treatment oftreatment treatment of treatment treatment Normal limits Erythrocytes,10¹²/l 2.9 4.0 4.3 4.1 4.4 4.0-5.0 Hemoglobin, g/l 118 129 134 130 132130.0-160.0 Platelets, 10⁹/l 178 228 242 204 218 180.0-320.0 Leukocytes,10⁹/l 12.4 8.2 5.4 5.6 4.8 4.0-9.0 Neutrophils and rods, % 10 5 4 4 31-6 Segmentonuclear 72 54 47 40 51 47-72 neutrophils, % Lymphocytes, % 832 39 45 36 19-37 Monocytes, % 3 5 6 8 6  3-11 Eosinophils, % 7 4 4 3 4 0-11 ESR, mm/h 48 18 13 14 15  2-10 Total protein, g/l 54 69 72 68 7065-85 ALT/mmol/hL 0.8 0.3 0.22 0.28 0.3 0.1-0.7 AST/mmol/hL 0.4 0.3 0.20.22 0.2 0.1-0.5 α-amylase, g/hL 112 33 28 26 20 12-32 LDH MU/l 1082 454352 178 212 <450 Lymphocytes 992 2624 2106 2520 1728 1200-3000B-lymphocytes (CD20+) 174 384 304 388 241 200-400 10⁶/l T-helpers(CD4+), 10⁶/l 514 825 932 926 834  700-1100 T-suppressors 102 584 784732 701 500-900 (CD8+) 10⁶/l CD4+/CD8+ ratio 5.0 1.41 1.19 1.27 1.191.0-1.5 CD25+ 154 286 356 482 476 208-576 NK-cells (CD16/56+) 172 196383 412 396 200-400 IgA, g/l 3.4 3.6 4.8 4.6 4.1 0.8-5.2 IgM, g/l 2.12.2 3.4 3.0 216 0.6-3.8 IgG, g/l 23.4 18.4 17.2 17.3 16.8  6.0-18.0Circulating immune 476 448 225 214 174  50-100 complexes

EXAMPLE 25 Therapeutical Effect of GSSG Series Preparations in Patientwith Severe Postoperative Complications

Diagnosis: Post-intubation scar stenosis of the trachea,tracheoesophageal fistula, post operative empyema of the right pleuralcavity.

A male 22 year old patient was admitted to the surgical division of theState Scientific Center of Pulmonology of the Ministry of Health ofRussia Federation in March 1996 after linear sent of the trachea wasplaced. After it was removed there was a relapse of the trachealstenosis and the signs of tracheoesophageal fistula appeared. Due tothis in April 1996 the operation was performed: circular resection ofthe trachea, closure of the TE fistula, omentoplastics.

During the postoperative period right-sided pleural empyema developed.Despite massive antibiotic therapy, detoxification and pleural cavitydrainage, the patient's state was very severe. The patient was pale,pulse 120/min, rhythmic, BP 90/50 mm Hg, breathing rate 28/min,slightest physical exertion caused dyspnea, t 38.8-39.6° C.

On chest X-ray: increased density of the left lung due to diffuseinfiltration (edema). Paracostally in the lateral areas along the ribcage and posteriorly in the interlobular fissures there was a lot offluid. Enlarged median shadow in the upper region.

Significant leukocytosis (30×10⁹/l), increased neutrophil count,increased ESR (58 mm/h), hypertransaminasemia (see Table 40).

Due to the severity of the patient's disease, pulmonary insufficiencyand absence of any alternative treatment the decision was made to usecompassionate treatment with lithium salt of GSSG (GSSG-Li₂) in acomplex therapy.

Right after the first injections of GSSG-Li₂ (0.01-0.5 mg/kg per day)there was a certain positive effect—decreased intoxication (decrease oftemperature to 37.3C), pulse 88/min, resolved pulmonary insufficiency.That positive effect due to the use of the treatment became strongerafter the following injections (0.01-0.5 mg/kg per day, intravenouslyfor 5 days).

After another week of treatment with GSSG-Li₂ (0.01-0.5 mg/kg/day)combined with antibiotics (Claforan 6 g/day, Ampicillin plus Oxacillin,2 g/day) the patient's state was satisfactory. No complains; pulse80/min, BP 115/70 mm Hg, breathing slightly decreased on the right, inall regions.

No local inflammation after thoracotomy. Granulating wound in the siteof posterior thoracotomy without secretions. Bronchoscopy showed wideanastomosis without granulation. Chest X-ray—normal.

After a month of treatment with GSSG-Li₂ (0.01-0.5 mg/kg every other dayIV and IM for 3 weeks) the patient's condition was satisfactory. Bloodparameters within normal limits (see Table 40).

Summarized effect: quick regression of the purulent process;potentiation of the effect of antibacterial therapy; beneficial bloodchanges; detoxification; restoration of laboratory parameters.

TABLE 40 Laboratory parameters in patient with severe postoperativecomplications After one After one Before week of month of Parametertreatment treatment treatment Erythrocytes, 10¹²/l 3.6 4.0 4.3Hemoglobin, g/l 118 128 132 Platelets, 10⁹/l 200 212 248 Leukocytes,10⁹/l 30.0 8.4 7.6 Neutrophils and rods, % 12 5 4 Segmentonuclearneutrophils, % 55 55 51 Lymphocytes, % 22 31 36 Monocytes, % 4 4 6Eosinophils, % 7 5 2 ESR, mm/h 58 12 10 ALT/mmol/hL 1.1 0.42 0.32AST/mmol/hL 0.6 0.32 0.28 Lymphocytes, 10⁶/l 6600 2604 2736B-lymphocytes (CD20+) 10⁶/l 682 319 324 T-helpers (CD4+), 10⁶/l 2188 8941112 T-suppressors (CD8+) 10⁶/l 2245 824 879 CD4+/CD8+ ratio 0.98 1.091.27 IgA, g/l 4.1 4.5 3.8 IgM, g/l 1.8 2.7 2.4 IgG, g/l 19.8 16.3 17.5Circulating immune complexes 174 95 108

EXAMPLE 26 Therapeutical Effect of GSSG Series Preparations in Patientswith Multiple Sclerosis

We examined and treated 19 patients with cerebrospinal form of Multiplesclerosis (MS) aged 23-52 (Table 41). All patients were admitted to thehospital during exacerbation. Diagnosis was verified according to therecommendations of the International association of MS studies (1992).There was a prevalence of patients with progressive MS with relapses. In5 patients length of relapse was one month; in 7—one to three months, in7—over three months.

TABLE 41 The number of 23-40 patients Total years Over 40 SevereModerate Mild Male 4 2 2 4 — — Female 15 9 6 12 2 1

The 90 days' course of treatment was carried clut in the following way:

1. GSSG-Na in 10% solution of S-adenosyl-methionine intravenously, oncea day for 10 days, daily dose—0.01-0.5 mg/kg

2. Two weeks' break

3. GSSG-Zn in 10% solution of S-adenosyl-methionine intravenously, everyother day for 20 days, daily dose 0.01-0.5 mg/kg

4. Two weeks' break

5. bis-methionil-GSSG in 5% solution of ascorbic acid (vitamin C)intravenously and intramuscularly, alternating routes of administration(one day each), daily dose 0.01-0.5 mg/kg for 30 days.

Patients' investigation was carried out before treatment, one monthafter the beginning of the treatment, the 3 and 6 months after thebeginning of the treatment. CD3+, CD4+, CD8+, CD20+ lymphocyte countswere measured in peripheral blood with immunofluorescent method usingmonoclonal antibodies; CD4+/CD8+ ratio was counted (Table 42). Cellularimmunity was assessed with the Reaction of Suppression of LeukocyteAdhesion in the presence of neuro-specific antigens: S-100 protein,neuronal membrane antigens, myelin basic protein (Table 43). 25 blooddonors and patients with radiculopathy were used as the control group.

Initial state of the immune system was characterized by decrease totalCD3+ count, significant decrease in CD4+ count, increase in CD8+ count,dysbalance between subpopulations of helpers and suppressors, increasein CD20+ count.

After the treatment there was an improvement in cellular immunityparameters—normalization of CD3+ count, CD4+ count, and CD8+(T-suppressors) count. In the same time there was a decrease Insensitization of lymphocytes to one or several of the brain tissueantigens. There also was a phenomenon of immunological inversion ofS-100 and myelin basic protein levels (Table 43).

Restoration of immune system was accompanied by resolution ofneurological symptoms in 84% of patients.

TABLE 42 Changes in mean values of T and B lymphocyte count in MSpatients during the treatment with GSSG series preparations. Patientsbefore After one After three After six Parameter Donors treatment monthmonths months CD3+ % 55.2 ± 1.8 45.22 ± 1.80 64.08 ± 3.28 63.18 ± 2.0270.78 ± 2.86 CD4+ % 36.0 ± 2.0 28.64 ± 1.22 33.18 ± 2.86 43.64 ± 1.1855.78 ± 2.12 CD8+ % 19.3 ± 2.2 16.76 ± 0.88 20.10 ± 3.16 24.26 ± 2.5626.34 ± 2.08 CD4/CD8  1.90 ± 0.24  1.72 ± 0.42  1.65 ± 0.24  1.79 ± 0.08 2.11 ± 0.10 CD20+ % 12.8 ± 1.8 18.78 ± 0.44 15.26 ± 0.64 15.32 ± 1.4414.44 ± 1.20

TABLE 43 Parameters of sensitization of blood lymphocytes of MS patientsto brain tissue antigens in Reaction of Suppression of Adhesion ofLeukocytes (%) Patients before After one After three After six Antigentreatment month months months S-100 54.20 72.12 66.46 42.10 Neuronalmembranes 56.32 44.64 41.24 32.56 antigen Myelin basic protein 68.3477.30 54.323 42.20

Endogenous level of IFN α and γ and TNF was measuried in patients' serumand cerebrospinal fluid (CSF) (Table 45). An increase in IFN-α duringpatients' treatment should be stressed. TNF level correlated withneurological disability, measured by Kurtzke score.

TABLE 44 Changes in cytokine level (pcg/ml) after induction by GSSGmedications in MS patients Patients before After three treatment Afterone month months After six months IFN-α, pcg/ml 0 202.26 ± 4.38 312.14 ±6.28 406.18 ± 4.66 IFN-γ, pcg/ml 36.18 ± 4.42  28.6 ± 4.82  24.52 ± 3.46 12.4 ± 6.22 TNF, pcg/ml 64.38 ± 8.64  52.42 ± 7.62  51.86 ± 4.32  51.46± 3.80

Humoral immunity was assessed by the following parameters: B-lymphocyteswere counted by measuring superficial immune globulins inimmunofluorescent test, IgA, IgM, IgG level were measured by radicalimmunodiffusion in gel, circulating immune complexes were precipitatedin polyethilenglycole (Table 46). Before treatment all patients hadsignificant increase in circulating immune complexes and IgM, anddecrease in IgG. IgG level was significantly lower than in the controlgroup.

TABLE 45 Changes in mean levels of immunoglobulins and circulatingimmune complexes in MS during the treatment course with GSSGmedications. Patients before After one After three After six ParameterDonors treatment month months months IgA, mE 3.2 1.3 1.4 1.6 1.4 IgM, mE1.8 5.4 4.6 3.5 3.8 IgG, mE 8.4 4.2 8.4 13.4  12.3 Circulating 52.1 ±6.8 142.2 ± 124.4 ± 107.4 ± 68.2 ± 5.4 immune 8.1 4.4 4.0 complexes,units of optical density

Neurological condition of all patients during this course of treatmenthave more or less improved, there was a decrease in motor index ofHauzer, and improvement in quality of life.

While specific embodiments of the invention have been shown anddescribed, many modifications are possible. For example, otheringredients which do not affect the action of GSSG its derivativesand/or both its extenders, and enhancers/modulators may be intermixedwith GSSG alone or in combination with its both extenders, and-enhancers/modulators for application to the body. The dosage forms canbe packaged in kit form along with syringe or applicator of any type andcan be sterilized. Preferably instructions for application to a specificdiseases are included in any kit, including the therapeutic agent. Weindicate below preferred applications of GSSG or/and its derivativeswith or without both extenders, and enhancers/modulators in doses from0.01 to 0.5 mg of GSSG base per kg of body weight for GSSG and its salts(0.01 to 1.0 mg per kg for the GSSG derivatives) per day for one or moredays, or spread over several days, intravenously, intramuscularly,intralymphatically, epicutaneously, or intracavitary for up to 6 monthsas has been found effective for the diseases noted below.

A prophylactic, therapeutic use of the methods and therapeutic agents ofthis invention can be made for immunodeficiency states where individualshave been exposed to radioactive and chemical affliction in cases ofaccidents such as nuclear disasters.

Where the both various extenders and various enhancers/modulators havebeen noted, other specific extenders which prolong the half life of theoxidized glutathione, or/and other specific enhancers/modulatorsaltering beneficially GSSG/derivative effects may be used. In somecases, one or more of the both different extenders and differentenhancers/modulators can be used in combination. Other salts than thosespecifically noted for GSSG can be used and GSSG derivatives can be thesame salt forms as GSSG or other physiologically safe andtherapeutically effective forms.

While the drug for parenteral use is preferably in solution form, insome cases colloidal suspensions and the like can be used. Similarly,topical application can include the use of pharmaceutically acceptableointments, creams and other bases such as petrolatum bases which do notinteract with the GSSG/derivatives, as well as with both extenders andenhancers/modulators. Such base materials are known in the art(petrolatum, lanolin, spermaceti; with inter alia addition ofacetylsalicilic acid).

Infectious and Immunopathology Diseases

All dosage rates given in the following section below are indicated forGSSG and its salts also referred to as “GSSG base”. Because “GSSG base”does not account for the weight of attached derivative chains to theGSSG base the corresponding dosage rates for the GSSG derivatives shouldbe within the interval of from the same as for GSSG base lower level, upto a level which is two-threefold as much as upper one for GSSG and itssalts.

AIDS: dosage rate—from 5 to 30 mg per day, entire course is of 6 monthduration, with 2 week break after each month;

the administration regimen during the first week—a single injectiondaily, alternating regimen: one day—intravenous injection, every otherday—intramuscular injection;

during the second week—twice a day: one time intravenously (in themorning), other time intramuscularly (in the evening);

the third and the forth weeks—three times a week: 1^(st)time—intravenous injection, 2^(nd) and 3^(rd) time—intramuscularinjection;

In case of encephalopathy it is recommended lumbar injections of themedicine once a week during three weeks.

Hepatitis: dosage rate—from 5 to 10 mg per day, entire course is from 1to 2 months;

during the first three weeks—a single injection daily, alternatingregimen: one day—intravenous injection, every other day—intramuscularinjection;

afterwards:—two or three injections per a week, 1^(st) time—intravenousinjection, 2^(nd) and 3^(rd) time

intramuscular injection;

Herpes: the medicine administration course is the same as in hepatitis.

Tuberculosis: Inactive phase: dosage rate—from 5 to 10 mg per day,entire course is of 6 months, with 2 week break after each month and 1month break after the 3 months of the medicine administration.

during the first three weeks—a single injection daily, alternatingregimen: one day—intravenous injection, every other day—intramuscularinjection;

during the forth week—two or three injections per a week, 1^(st)time—intravenous injection, 2^(nd) and 3^(rd) time—intramuscularinjection;

Active phase: dosage rate—from 5 to 10 mg per day, entire course is of 6months, the administration regimen in an active phase is the same as ininactive phase.

Meningitis: dosage rate—from 5 to 90 mg per day, entire course is of 2months;

during the first two weeks—twice a day: one time intravenously (in themorning), other time intramuscularly (in the evening);

afterwards:—two or three injections per a week, 1^(st) time—intravenousinjection, 2^(nd) and 3^(rd) time

intramuscular injection;

Lumbar injections of the medicine—a single injection daily isrecommended during three days.

Peritonitis: the medicine administration course is the same as inmeningitis (except for lumbar injections).

Sepsis: dosage rate—from 5 to 60 mg per day, entire course is no lessthan 1 months up to full normalization of clinical state and blood data;

during the first two or tree weeks—twice a day: one time intravenously(in the morning), other time intramuscularly (in the evening);

afterwards:—two or three injections per a week, 1^(st) time—intravenousinjection, 2^(nd) and 3^(rd) time—intramuscular injection;

Purulent post-operative infectious complications—the medicineadministration course is the same as in sepsis.

Immunodepression: dosage rate—from 5 to 20 mg per day, entire course isof 6 months, with 2 week break after each month;

during the first three weeks—a single injection daily, alternatingregimen: one day—intravenous injection, every other day—intramuscularinjection;

during the forth week—two or three injections per a week, 1^(st)time—intravenous injection, 2^(nd) and 3^(rd) time—intramuscularinjection;

Immunodeficiencies of infectious, radiation, or toxic origin: dosagerate is from 5 to 30 mg per day; the medicine administration course isthe same as in immunodepression.

Multiple sclerosis: dosage rate is from 5 to 20 mg per day, entirecourse is of 3 months with 2 week break after each month, and after 6month break—the repeating of entire course;

the 1^(st) month of the entire course: a single injection daily,alternating regimen: one day—intravenous injection, every otherday—intramuscular injection;

the 2^(nd) month of the entire course: three injections per a week, 1 sttime—intravenous injection, 2nd and 3rd time—intramuscular injection;

the 3^(rd) month of the entire course: two intramuscular injections pera week, with dosage rate from 5 to 10 mg.

Neurodegenerative diseases: the medicine administration course is thesame as in multiple sclerosis.

Alzheimer's sclerosis: the medicine administration course is the same asin multiple sclerosis.

Amyotrophic lateral sclerosis: the medicine administration course is thesame as in multiple sclerosis.

Glomerulonephritis: dosage rate is from 5 to 30 mg per day, entirecourse is from 1 to 3 months with 2 week break after each month;

during the first two weeks—a single injection daily, alternatingregimen: one day—intravenous injection, every other day—intramuscularinjection;

afterwards:—two or three injections per a week, 1^(st) time—intravenousinjection, 2^(nd) and 3^(rd) time

intramuscular injection;

Collagenoses:—the medicine administration course is the same as inglomerulonephritis.

Rheumatoid arthritis:—the medicine administration course is the same asin Glomerulonephritis.

Systemic lupus erythematosus:—the medicine administration course is thesame as in Glomerulonephritis.

Allergic diseases:—the medicine administration course is the same as inGlomerulonephritis, along with the topical application of ointment (1-3%containing the active compound)—during 2 weeks, a'single application perday, afterwards:—two applications per a week.

Psoriasis:—the medicine administration course is the same as inGlomerulonephritis, along with the topical application of ointment (1-3%containing of the active compound)—during 2 weeks, a single applicationper day, afterwards:—two applications per a week.

Neoplasms: dosage rate is from 5 to 90 mg per day, entire course is from1 to 6 months with 2-4 week break after each month;

a single injection daily, alternating regimen: one day—intravenousinjection, every other day—intramuscular injection, without or alongwith endolymphatic application (dosage rate—from 30-90 mg per day)during 10 days, and local application (regional perfusion) bycatheterization (dosage rate—from 30-90 mg per day) during 7 days, threeor four applications per week;

the treatment scheme is recommended along with polychemiotherapeutictreatment.

Metastatic processes and hemoblastoses: dosage rate is from 5 to 90 mgper day, entire course is from 1 to 6 months with 2-4 week break aftereach month;

a single injection daily, alternating regimen: one day—intravenousinjection, every other day—intramuscular injection, along with localapplication (regional perfusion) by catheterization (dosage rate—from30-90 mg per day) during 7 days, three or four applications per week.

Lympho proliferative diseases (Lymphogranulomatosis and Lymphoma):dosage rate is from 5 to 90 mg per day, entire course is from 1 to 6months with 2-4 week break after each month;

during the first three weeks—a single injection daily, alternatingregimen: one day—intravenous injection, every other day—intramuscularinjection, along with endolymphatic application (dosage rate—from 30-90mg per day) during first 10 days; afterwards:—the same treatment schemein combination with glucocorticoides and cytostatics.

FOOTNOTES

1. Holmlund J. T. Cytokines. Cancer Chemother Biol Response Modif. 1993.14 P 150-206.

2. Hansson M., Soderstrom T. The colony stimulating factors. Med OncolTumor Pharmacother. 1993. 10(1-2). P 5-12.

3. Dillman R. O. The clinical experience with interleukin-2 in cancertherapy. Cancer Biother. 1994 Fal. 9(3). P 183-209.

4. Whittington R., Faulds D. Interleukin-2. A review of itspharmacological properties and therapeutic use in patients with cancer.Drugs. September 1993 46(3). P 446-514.

5. Hieber U., Heim M E. Tumor necrosis factor for the treatment ofmalignancies. Oncology. March-April. 1994 51(2). P 142-53.

6. Morstyn G., Sheridan W. P. Hematopoietic growth factors in cancerchemotherapy. Cancer Chemother Biol Response. Modif. 1993. 14 P 353-70.

7. Neidhart J. A. Hematopoietic cytokines. Current use in cancertherapy. Cancer. December 1. 1993 72(11 Suppl). P 3381-6.

8. Murray H. W. Interferon-gamma and host antimicrobial defense: currentand future clinical applications. Am J Med. November 1994 97(5). P459-67.

9. Cirelli R. Tyring S. K. Interferons in human papillomavirusinfections. Antiviral Res. July 1994 24(2-3). P 191-204.

10. Sher A. Coffman R. L. Regulation of immunity to parasites by T-cellsand T-cell derived cytokines. Annu. Rev. Immunol., 1992, 10. P. 385-409.

11. Gillan E., Plunkett M., Cairo M. S. Colony-stimulating factors inthe modulation of sepsis. New Horiz. February 1993 1 (1). P 96-109.

12. Nelson S. Role of granulocyte colony-stimulating factor in theimmune response to acute bacterial infection in the nonneutropenic host:an overview. Clin Infect Dis. February 1994 18 Suppl 2 P S197-204.

13. Offenstadt G., Guidet B., Staikowsky F. Cytokines and severeinfections. Pathol Biol (Paris). October 1993 41(8 Pt 2). P 820-31.

14. Nemunaitis J. Use of hematopoietic growth factors in marrowtransplantation. Curr Opin Oncol. March 1994 6(2). P 139-45.

15. Mittelman M., Lessin L. S. Clinical application of recombinanterythropoietin in myelodysplasia. Hematol Oncol Clin North Am. October1994 8(5). P 993-1009.

16. Forman A. D. Neurologic complications of cytokine therapy. Oncology(Huntingt). April 1994 8(4). P 105-10; discussion 113, 116-7.

17. Hack C. E., Ogilvie A. C., Eisele B., Eerenberg A. J., Wagstaff J.,Thijs L. G. C1-inhibitor substitution therapy in septic shock and in thevascular leak syndrome induced by high doses of interleukin-2. IntensiveCare Med. 1993. 19 Suppl 1 PSI19-28.

18. Hieber U., Heim M. E. Tumor necrosis factor for the treatment ofmalignancies. Oncology. March-April 1994 51(2). P 142-53.

19. Saito M. OK-432, a killed streptococcal preparation, in thetreatment of animal and human cancer and its mechanisms of action. Formon immunomodulators. Ed. Guenounou M. John Libbey Eurotext, Paris, 1995,P 1-11.

20. Barot-Ciorbaru R., Bona C. Immunomodulators from Nocardia opaca.Form on immunomodulators. Ed. Guenounou M. John Libbey Eurotext, Paris,1995, P 1-11.

21. Bloy C. Morales M., Gu enounou M. RU 41740 (Biostim), animmunomodulating agent from bacterial origin. Ed. Guenounou M. JohnLibbey Eurotext, Paris, 1995, P 1-11.

22. Meister A. Anderson M. E. Glutathione. Annu. Rev. Biochem., 1983,52:711-60.

23. Beutler E. Nutritional and metabolic aspects of glutathione. Review.Annu. Rev. Nutr., 1989, 9:287.

24. Textbook of biochemistry: with clinical correlations. Ed. Devlin T.M., 3rd ed. 1992, Wiley-Liss, Inc., N.Y. P 522-525.

25. Ehrer J. P., Lund L. G. Cellular reducing equivalents and oxidativestress. Free Radic Biol Med. July. 1994 17(1). P 65-75.

26. Michiels C., Raes M., Toussaint O., Remacle J. Importance ofSe-glutathione peroxidase, catalase, and Cu/Zn-SOD for cell survivalagainst oxidative stress. Free Radic Biol Med. September 1994 17(3). P235-48.

27. Cohen G. Enzymatic/nonenzymatic sources of oxyradicals andregulation of anti6xidant defenses. Ann N.Y. Acad Sci. Nov. 17, 1994738. P 8-14.

28. Beckett G. J., Hayes J. D. Glutathione S-transferase: biomedicalapplications. Advan. Clin. Chem. 1993, vol. 30, P 281-380.

29. Composition and method for disease treatment. PCT/US/92/04653.

30. Droge W., Schultze-Osthoff K., Mihm S., Galter D., Schenk H., Eck H.P., Roth S., Gmunder H. Functions of glutathione and glutathionedisulfide in immunology and immunopathology. FASEB J. November 19948(14). P 1131-8.

31. Sardesai V., M. Role of antioxidants in health maintenance. NutrClin Pract. February 1995 10(1). P 19-25.

32. Giugliano D., Ceriello A., Paoliso G. Diabetes mellitus,hypertension, and cardiovascular disease: which role for oxidativestress? Metabolism. March 1995 44(3). P 363-8.

33. Keusch G. T. Antioxidants in infection. J Nutr Sci Vitaminol(Tokyo). 1993. 39 Suppl P S 23-33.

34. Dipeptide compound having pharmaceutical activity and compositionscontaining them. U.S. Pat. No. 4,761,399.

35. G-L-Glutamyl-L-cysteine ethyl ester and pharmaceutical compositionscontaining the same as an effective ingredient. U.S. Pat. No. 4,927,808.

36. Therapeutic agents for ischemic heart diseases. U.S. Pat. No.4,968,671.

37. Method for insuring adequate intracellular glutathione in tissue. EP0 502 313 A2.

38. Composition and method for disease treatment. PCT/US/92/04653.

39. Glutathione as hemoprotective agent. PCT/EP/93/01494.

40. Pharmaceutical compositions having antineoplastic activity. U.S.Pat. No. 4,871,528.

41. Sokolovsky M., Wilchek M. Patchornik A. On the synthesis of cysteinpeptides. J. Amer. Chem. Soc. March 1964, 86(6), P 1202-6.

What is claimed is:
 1. A method of treating a mammalian body comprisingintroducing to a mammalian body an effective amount to stimulateproduction of cytokines or hemopoietic factors or both of an oxidizedglutathione form selected from the group consisting of oxidizedglutathione, a pharmaceutically acceptable oxidized glutathione salt, apharmaceutically acceptable glutathione derivative and mixtures thereof,along with an extender of the half-life of the oxidized glutathione formselected from the group consisting of dimethyl sulfoxide, inosine andcystamine, and an enhancer/beneficial modifier of a therapeutic effectof the oxidized glutathione form, for a period of time to obtain adesired therapeutic effect.
 2. The method of claim 1, wherein theextender is dimethyl sulfoxide.
 3. The method of claim 1, wherein theextender is inosine.
 4. The method of claim 1, wherein the extender iscystamine.
 5. A method of treating a mammalian body comprisingintroducing to a mammalian body an effective amount to stimulateproduction of cytokines or hemopoietic factors or both of an oxidizedglutathione form selected from the group consisting of oxidizedglutathione, a pharmaceutically acceptable oxidized glutathione salt, apharmaceutically acceptable glutathione derivative and mixtures thereof,along with an extender of the half-life of the oxidized glutathioneform, and an enhancer/beneficial modifier of a therapeutic effect of theoxidized glutathione form selected from the group consisting ofcholine-chloride, S-adenosyl-methionine and lipoic acid, for a period oftime to obtain a desired therapeutic effect.
 6. The method of claim 5,wherein the enhancer/beneficial modifier is choline-chloride.
 7. Themethod of claim 5, wherein said enhancer/beneficial modifier isS-adenosyl-methionine.
 8. The method of claim 5, wherein saidenhancer/beneficial modifier is lipoic acid.
 9. A method of treating amammalian body comprising introducing to a mammalian body an effectiveamount to stimulate production of cytokines or hemopoietic factors orboth of an oxidized glutathione form that is a pharmaceuticallyacceptable oxidized glutathione salt selected from the group consistingof a dilithium salt, a salt that contains one or more atoms ofpotassium, a salt that contains one or more atoms of calcium, a saltthat contains one or more atoms of zinc, a salt that contains one ormore atoms of molybdenum, a salt that contains one or more atoms ofvanadium, and a salt that contains one or more atoms of fluoride, andmixtures thereof, along with an extender of the half-life of theoxidized glutathione form, and an enhancer/beneficial modifier of atherapeutic effect of the oxidized glutathione form, for a period oftime to obtain a desired therapeutic effect.
 10. The method of claim 9,wherein the glutathione salt is a dilithium salt.
 11. The method ofclaim 9, wherein the glutathione salt is a salt that contains one ormore atoms of potassium.
 12. The method of claim 9, wherein theglutathione salt is a salt that contains one or more atoms of calcium.13. The method of claim 9, wherein the glutathione salt is a salt thatcontains one or more atoms of zinc.
 14. The method of claim 9, whereinthe glutathione salt is a salt that contains one or more atoms ofmolybdenum.
 15. The method of claim 9, wherein the glutathione salt is asalt that contains one or more atoms of vanadium.
 16. The method ofclaim 9, wherein the glutathione salt is a salt that contains one ormore atoms of fluoride.
 17. A method of treating a mammalian bodycomprising introducing to a mammalian body an effective amount tostimulate production of cytokines or hemopoietic factors or both of anoxidized glutathione form that is a pharmaceutically acceptableglutathione derivative selected from the group consisting of GSSGcovalently bound to cysteamine (S-thioethylamine-glutathione disulfide),GSSG covalently bound to lipoic acid (bis-[6,8-thiooctanyl]•glutathionedisulfide), GSSG covalently bound to a member of the group consisting ofcarnosine ([β-alanyl-histidyl]•glutathione disulfide) and adenosine([9-β-D-ribofiuranosyladenyl]•glutathione disulfide), and GSSGcovalently bound to methionine(bis-[2-amino-4-[methylthio]butanoyl]•glutathione disulfide) andmixtures thereof, along with an extender of the half-life of theoxidized glutathione form, and an enhance/beneficial modifier of atherapeutic effect of the oxidized glutathione form, for a period oftime to obtain a desired therapeutic effect.
 18. The method of claim 17,wherein the glutathione derivative is GSSG covalently bound tocysteamine (S-thioethylaniine-glutathione disulfide).
 19. The method ofclaim 17, wherein the glutathione derivative is GSSG covalently bound tolipoic acid (bis-[6,8-thiooctanyl]•glutathione disulfide).
 20. Themethod of claim 17, wherein the glutathione derivative is GSSGcovalently bound to a member of the group consisting of carnosine([β-alanyl-histidyl]•glutathione disulfide) and adenosine([9β-D-ribofuranosyladenyl]•glutathione disulfide).
 21. The method ofclaim 17, wherein the glutathione derivative is GSSG covalently bound tomethionine (bis-[2-amino-4-[methylthio]butanoyl]•glutathione disulfide).22. A therapeutic agent for treating neoplastic, infectious,hematologic, immunologic or other diseases, said therapeutic agentcomprising oxidized glutathione, and/or a pharmaceutically acceptableglutathione salt, and/or a pharmaceutically acceptable glutathionederivative, in an effective amount to stimulate production of cytokinesand hemopoietic factors or both, to obtain-a desired therapeutic-effect,along with a pharmaceutically acceptable extender capable of enhancingand prolonging a therapeutic effect of the therapeutic-agent byincreasing the half-life of the therapeutic agent, selected from thegroup consisting of hydrogen peroxide, dimethyl sulfoxide, inosine andcystamine, wherein the therapeutic agent is in a pharmaceuticallyacceptable excipient and is formulated in the form of a sterileinjectable solution of oxidized glutathione, and/or a pharmaceuticallyacceptable glutathione salt, and/or a pharmaceutically acceptableglutathione derivative.
 23. The therapeutic agent of claim 22, whereinthe extender is hydrogen peroxide.
 24. The therapeutic agent of claim22, wherein the extender is dimethyl sulfoxide.
 25. The therapeuticagent of claim 22, wherein the extender is inosine.
 26. The therapeuticagent of claim 22, wherein the extender is cystamine.
 27. A therapeuticagent for treating neoplastic, infectious, hematologic, immunologic orother diseases, said therapeutic agent comprising oxidized glutathione,and/or a pharmaceutically acceptable glutathione salt, and/or apharmaceutically acceptable glutathione derivative, in an effectiveamount to stimulate production of cytokines and hemopoietic factors orboth, to obtain a desiredtherapeutic effect, along with apharmaceutically acceptable extender capable of enhancing and prolonginga therapeutic effect of the therapeutic agent by increasing thehalf-life of the therapeutic agent, and a pharmaceutically acceptableenhancer/beneficial modifier capable of enhancing and/or alteringbeneficially a therapeutic effect of the therapeutic agent by mechanismsother than increasing the half-life of the therapeutic agent, andselected from the group consisting of choline-chloride,S-adenosyl-methionine and lipoic acid, wherein the therapeuptic agent isin a pharmaceutically acceptable excipient and is formulated in the formof a sterile injectable solution of oxidized glutathione, and/or apharmaceutically acceptable glutathione salt, and/or a pharmaceuticallyacceptable glutathione derivative.
 28. The therapeutic agent of claim27, wherein the enhancer/beneficial modifier is choline-chloride. 29.The therapeutic agent of claim 27, wherein the enhancer/beneficialmodifier is S-adenosyl-methionine.
 30. The therapeutic agent of claim27, wherein the enhancer/beneficial modifier is lipoic acid.
 31. Themethod of claims 1, 5, 9, 17, wherein the oxidized glutathione form isintroduced parenterally.
 32. The method of claims 1, 5, 9, or 17,wherein the oxidized glutathione form is introduced topically.
 33. Themethod of claims 1, 5, 9, or 17, wherein the mammalian body is in needof stimulation of cytokine or hemopoietic factor production to treat acondition selected from the group consisting of neoplastic, infectious,hematologic, immunologic and other diseases.
 34. The method of claim 33,wherein the disease is an infectious disease.
 35. The method of claim33, herein the disease is a hematologic disease.
 36. The method of claim33, wherein the disease is an immunologic disease.
 37. The method ofclaim 33, wherein said disease is a neoplastic disease.
 38. The methodof claim 33, wherein the disease is selected from the group consistingof AIDS, hepatitis, herpes, tuberculosis, meningitis, peritonitis,sepsis, and purulent post-operative complications caused by infection.39. The method of claim 33, wherein said disease is selected from thegroup consisting of immunodepression, multiple sclerosis, Alzheimer'ssclerosis, neurodegenerative diseases, amyotrophic lateral sclerosis,glomerulonephritis, collagenosis, rheumatoid arthritis, lupus,psoriasis, diabetes mellitus and allergic disease.
 40. The method ofclaim 33, wherein the disease is selected from the group consisting ofmetastatic spreading, hemoblastosis, malignant tumors,lymphogranulomatosis, lymphomas, and immunodeficiency caused byradioactive or chemical affliction.